Methods for differentiating neural stem cells to neurons or smooth muscle cells using TGT-β super family growth factors

ABSTRACT

Method for producing a population of mammalian neurons and/or smooth muscle cells comprising contacting at least one mammalian neural stem cell with a culture medium containing one or more growth factors from the TGF- beta  super family and detecting the differentiation of stem cell to a population of neurons or smooth muscle cells.

This is a continuation in part of U.S. patent application Ser. No.08/188,286 filed Jan. 28, 1994, now U.S. Pat. No. 5,654,183, which is acontinuation-in-part of PCT Application No. PCT/US93/07000 filed Jul.26, 1993, published Feb. 3, 1994, as WO 94/02593, which is acontinuation-in-part of U.S. patent application Ser. No. 07/969,088filed Oct. 29, 1992, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 07/920,617, filed Jul. 27, 1992, nowabandoned. This application also claims benefit under 35 U.S.C. § 119(e)U.S. provisional patent application No. 60/044,797, filed Apr. 24, 1997.

FIELD OF THE INVENTION

The invention relates to methods for the differentiation of mammalianmultipotent neural stem cells to neurons or smooth muscle cells.

BACKGROUND

The neural crest is a transient embryonic precursor population, whosederivatives include cells having widely different morphologies,characteristics and functions. These derivatives include the neurons andglia of the entire peripheral nervous system, melanocytes, cartilage andconnective tissue of the head and neck, stroma of various secretoryglands and cells in the outflow tract of the heart (for review, seeAnderson, D. J. (1989) Neuron 3:1-12). The neural crest generates mostof the peripheral nervous system (PNS), skin melanocytes, andmesectodermal derivatives such as smooth muscle (SM) cells, bone, andcartilage Horstadius, S., London; Oxford University Press (1950); LeDouarin et al. (1994) Curr. Opin. Genet. Devel. 4:685-695; Shah etal.(1996) Cell 85:331-343. Much of the knowledge of the developmentalpotential and fate of neural crest cells comes from studies in aviansystems. Fate maps have been established in aves and provide evidencethat several different crest cell derivatives may originate from thesame position along the neural tube (Le Dourain, N. M. (1980) Nature286:663-669). Schwann cells, melanocytes and sensory and sympatheticneurons can all derive from the truncal region of the neural tube. Onthe other hand, some derivatives were found to originate from specificregions of the crest, e.g., enteric ganglia from the vagal and sacralregions. These studies also revealed that the developmental potential ofthe neural crest population at a given location along the neural tube isgreater than its developmental fate. This suggests that the newenvironment encountered by the migrating crest cells influences theirdevelopmental fate.

Single-cell lineage analysis in vivo, as well as clonal analysis invitro, have reportedly shown that early avian neural crest cells aremultipotential during, or shortly after, their detachment and migrationfrom the neural tube. In avian systems, certain clones derived fromsingle neural crest cells in culture were reported to contain bothcatecholaminergic and pigmented cells (Sieber-Blum, M. et al. (1980)Dev. Biol. 80:96-106). Baroffio, A. et al. (1988) Proc. Natl. Acad. Sci.USA 85:5325-5329, reported that avian neural crest cells from thecephalic region could generate clones which gave rise to highlyheterogeneous progeny when grown on growth-arrested fibroblast feedercell layers.

In vivo demonstration of the multipotency of early neural crest cellswas reported in chickens by Bronner-Fraser, M. et al. (1989) Neuron3:755-766. Individual neural crest cells, prior to their migration fromthe neural tube, were injected with a fluorescent dye. After 48 hours,the clonal progeny of injected cells were found to reside in many or allof the locations to which neural crest cells migrate, including sensoryand sympathetic ganglia, peripheral motor nerves and the skin.Phenotypic analysis of the labeled cells revealed that at least someneural crest cells are multipotent in vivo.

Following migration from the neural tube, these early multipotent crestcells become segregated into different sublineages, which generaterestricted subsets of differentiated derivatives. The mechanisms wherebyneural crest cells become restricted to the various sublineages arepoorly understood. The fate of neural crest derivatives is known to becontrolled in some way by the embryonic location in which theirprecursors come to reside (Le Douarin, N. M. (1982) The Neural Crest.,Cambridge University Press, Cambridge, UK). The mechanism ofspecification for neural crest cells derivatives is not known. Inculture studies described above, investigators reported that clonesderived from primary neural crest cells exhibited a mixture ofphenotypes (Sieber-Blum, M. et al. (1980) ibid; Baroffio, A. et al.(1988) ibid; Cohen, A. M. et al. (1975) Dev. Biol. 46:262-280; Dupin, E.et al. (1990) Proc. Natl. Acad. Sci. USA 87:1119-1123). Some clonescontained only one differentiated cell type whereas other clonescontained many or all of the assayable crest phenotypes.

The observation that apparently committed progenitors and multipotentcells coexist in the neural crest may be interpreted to reflect apre-existing heterogeneity in the population of primary crest cells orit may reflect asynchrony in a population of cells that undergoes aprogressive restriction in developmental potential.

How growth factors influence the fate of multipotent progenitor cells isnot well understood. Most hematopoietic growth factors act selectivelyas survival factors, rather than instructively as lineage determinationsignals. Shah et al.(1996) Cell 85:331-343.

Given the uncertainty in the art concerning the developmental potentialof neural crest cells, it is apparent that a need exists for theisolation of neural crest cells in clonal cultures. Although culturesystems have been established which allow the growth and differentiationof isolated avian neural crest cells thereby permitting phenotypicidentification of their progeny, culture conditions which allow theself-renewal of multipotent mammalian neural crest cells have not beenreported. Such culture conditions are essential for the isolation ofmammalian neural crest stem cells. Such stem cells are necessary inorder to understand how multipotent neural crest cells become restrictedto the various neural crest derivatives. In particular, cultureconditions which allow the growth and self-renewal of mammalian neuralcrest stem cells are desirable so that the particulars of thedevelopment of these mammalian stem cells may be ascertained. This isdesirable because a number of tumors of neural crest derivatives existin mammals, particularly humans. Knowledge of mammalian neural creststem cell development is therefore needed to understand these disordersin humans. Additionally, the ability to isolate and grow mammalianneural crest stem cells in vitro allows for the possibility of usingsaid stem cells to treat peripheral neurological disorders in mammals,particularly humans. The ability to preferentially differentiate neuralstem cells allows for a number of treatments which require the growth orregeneration of damaged, injured or deficient neurons or smooth musclecells.

It is therefore an object of the invention to provide methods utilizinggrowth factors from the TGF-β superfamily to preferentiallydifferentiate neural stem cells to neurons and/or smooth muscle cells.

It is a further object of the invention to utilize growth factors fromthe TGF-β superfamily to preferentially increase the population ofmammalian neurons and/or smooth muscle cells.

It is also an object of the invention to remove cells from a patient,treat the cells with growth factors from the TGF-β superfamily in orderto preferentially increase the population of mammalian neurons and/orsmooth muscle cells, and return the cells to the patient.

It is another object of the invention to treat a patient with growthfactors from the TGF-β superfamily in order to preferentially increasethe population of mammalian neurons and/or smooth muscle cells withinsaid patient.

It is an additional object of the invention to introduce foreign(heterologous) nucleic acid into multipotent neural stem cells or theirprogeny and utilize growth factors from the TGF-β superfamily topreferentially differentiate such transformed neural stem cells toneurons and/or smooth muscle cells.

SUMMARY OF THE INVENTION

In accordance with the forgoing objects, the invention includes a methodfor producing a population of mammalian neurons and/or smooth musclecells comprising contacting at least one mammalian neural stem cell witha culture medium containing one or more growth factors from the TGF-βsuperfamily. The growth factor may be contained within an extract frommammalian tissue or may be substantially pure. The growth factor orcombination of growth factors may administered to cells in vivo or exvivo.

The growth factors include the group of biologically active polypeptideswhich control the growth and differentiation of responsive cells.Examples of these growth factors include but are not limited to; theTGF-β series of growth factors as exemplified by TGF-β1-TGF-β4, the BMPseries of growth factors as exemplified by BMP-2 and BMP-4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the migration of rat neural crest cells from the neuraltube.

FIG. 1B demonstrates the expression of LNGFR and nestin by neural crestcells.

FIGS. 1C and 1D show the FACS profile from neural crest cells stainedwith anti-LNGFR (1D) and a control showing the background staining ofthe secondary antibody (1C).

FIG. 2 demonstrates the clonal expansion of LNGFR⁺, nestin⁺ rat neuralcrest cells.

FIG. 3 is a flow chart summarizing experiments demonstrating themultipotency of mammalian neural crest cells.

FIG. 4 demonstrates the expression of neuronal traits in clones derivedfrom LNGFR⁺ founder cells.

FIG. 5 demonstrates the expression of Schwann cell phenotype by neuralcrest-derived glia.

FIG. 6 shows the expression of peripherin, GFAP, and O₄ in a clonederived from a LNGFR⁺ founder cell.

FIG. 7 is a flow chart summarizing experiments demonstrating theselfrenewal of mammalian neural crest cells.

FIG. 8 demonstrates the self-renewal of multipotent neural crest cells.

FIG. 9 demonstrates the multipotency of secondary founder cells.

FIG. 10 provides a flow chart summarizing experiments demonstrating thesubstrate effect on the fate of mammalian neural crest cells.

FIG. 11 demonstrates that the neuronal differentiation of multipotentneural crest cells is affected by their substrate.

FIG. 12 summarizes the percentage of different clone types which resultwhen founder cells are grown on either FN or FN/PDL substrates.

FIG. 13 provides a flow chart summarizing experiments demonstrating theinstructive effect of the substrate on neural crest cell fate.

FIG. 14 summarizes the percentage of the different clone types whichresult when founder cells are treated with a PDL lysine overlay at 48hours (panel A) or day 5 (panel B).

FIG. 15 demonstrates the genetic-engineering of a multipotent neuralstem cell. Panel A depicts the expression of E. coli β-galactosidase(lacZ) in neural crest stem cells following infection with alacZ-containing retrovirus. β-galactosidase⁺ cells are indicated by thesolid arrows. Panel B depicts neural crest stem cells in phase contrast,in the same microscopic field as shown in Panel A. Cells which do notexpress β-galactosidase are indicated by open arrows.

FIG. 16 demonstrates the specificity of a supernatant from a hybridomaculture producing monoclonal antibody specific to mouse LNGFR.Supernatants were screened using live Schwann cells isolated from mousesciatic nerve. Panel A shows that most cells are stained with anti-LNGFRantibody (red staining; open arrows). Panel B shows Schwann cell nucleicounter stained with DAPI. Comparison with Panel A reveals a few cellsnot labeled by anti-LNGFR antibody (blue staining; open arrows).

FIGS. 17 A and B depict the identification of smooth muscle cells inneural crest cultures. Cultures of neural crest stem cells were fixedand double-labeled with antibodies to p75-LNGFR (Panel B, orangestaining), and SMA (Panel B, green staining). The cultures were alsolabeled with DAPI, a nuclear dye (Panel B, blue ovals). A phase contrastimage of the microscopic field is shown in FIG. 17A. Note that the p75+cells (Panel B, solid arrow) do not express SMA<whereas the SMA+ cells(Panel B, open arrows) do not express p75.

FIGS. 18 A and B demonstrate that individual neural cres cells cangenerate neurons, glia and smooth muscle cells. The figures illustratethree views of a clone derived from a single p75+ neural crest foundercell, grown for two weeks in standard medium. A neuron is identifiablein the clone by virtue of peripherin expression (Panel B, arrowhead) andlong neurites (Panel A). Glia are identifiable by GFAP expression (PanelC, orange staining, open arrows), and a smooth muscle cell is identifiedby staining with anti-SMA (Panel C, green staining, closed arrow).Nuclei of all cells have been labeled blue with DAPI (Panel C).

FIGS. 19 A, B and C demonstrate that smooth muscle cell differentiationis promoted by fetal bovine serum. Shown are three views of a colony ofneural crest cells grown in 5% fetal bovine serum. These cells do notexpress p75-LNGFR under these conditions. Cells visible byphase-contrast (Panel A) express both SMA (Panel B, red staining) andalso desmin (Panel C, green staining).

FIGS. 20 A and B demonstrate that neural crest-derived smooth musclecells express calponin. The culture is similar to that in FIG. 19,except the cells were doubly-labeled with anti-SMA (Panel B, redstaining) and calponin (Panel B, green staining). Cells that co-expressboth markers stain orange due to blending of the two colors (Panel B).

FIG. 21 demonstrates that bone marrow phogenic protein 2 ("BMP2")induces autonomic neuronal differentiation of NCSCs. NCSCs were culturedin rBMP2 (Panels A-D) or control medium (Panels E and F), fixed andimmunostained with antibody to peripherin (Panels B and F) or monoclonalantibody B2 (Panel D) followed by phycoerythrin conjugated secondantibody and DAPI counterstain. Phase contrast views presented by way ofcomparison indicate neuronal phase-bright colonies in Panel A, which areperipherin+ (Panel B)in contrast to the colonies in Panels E and F whichare peripherin-resemble undifferentiated neurons.

FIG. 22 demonstrates that recombinant BMP2 or TGFβ inducedifferentiation of NCSCs to smooth muscle. NCSCs were cultured inrBMP2(Panels A-C) or rTGFβ (Panels D-F), fixed and triply-labeled withperipherin (second antibody bound to HRP), anti-SMA (green staining) andcalponin (red staining). Cells that co-express both SMA ans calponinstain counterstain. Phase contrast views are presented for purposes ofcomparison in Panels A and D. 25% of colonies in rBMP-2 contained SM andneurons (Panels A-C) while 99% of colonies in contained SM-like cells(Panels D-F).

FIG. 23 demonstrates that recombinant BMP2 and TGF-β induce distinctresponses over a wide range of doses. Cells cultured in rBMP2(Panels Aand B) or rTGF-β (Panel C) were fixed 4 days after the addition offactors, stained for the markers indicated, and the proportion ofdifferent colony types determined. In rBMP2, both neuron-only andSM-only colonies are obtained at essentially all doses. (Panels A and B)In rTGF-β, SM-only colonies are evident in a dose-dependent manner(Panel C). At no concentration of TGFβ1 were neurons observed (data notshown, see text Shah et al., 1996).

FIG. 24 demonstrates instructive effects of BMPβ and TGFβ based onserial observation of identified clones. Individual founder cells wereidentified and photographed at day 0 (DO) in control medium, and thenrBMPS (Panel B) or rTGFβ1 (Panel C) was added to some plates whileothers were maintained as controls (Panel A). The same clones werephotographed every 24 hr for the next 4 days (D1, D2, etc.) In rBMP2([Panel B] column), the founder cell divides and all of its progenybecome neurons, whereas in rTGFβ1 ([Panel C] column), all the foundercell progeny become SM-like cells. No dying cells or cell carcasses areobserved in any of the developing clones.

FIG. 25 demonstrate that BMP2 induces MASH1 expression in NCSCs based onlabeling with an anti-MASH1 monoclonal antibody. NCSCs were grown atclonal density in rBMP2 (Panels A and B) or in control medium (Panels Cand D) for 12 hr, then fixed and labeled with anti-MASH1 monoclonalantibody. At 12 hours, ˜85% of the colonies cultures in BMP2 were MASH1+(Panels A and B) as contrasted to those grown in control medium (PanelsC and D) wherein very few NCSCs were MASH1+.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed, in part, to the isolation and clonalpropagation of non-transformed mammalian neural crest stem cells and tomultipotent neural stem cells from other embryonic and adult tissue. Theinvention also includes the production of neural crest stem cell andmultipotent neural stem cell derivatives including progenitor and moredifferentiated cells of the neuronal and smooth muscle lineages. Theinvention is illustrated using neural crest stem cells isolated from therat. The invention, however, encompasses all mammalian neural crest stemcells and multipotent neural stem cells and their derivatives and is notlimited to neural crest stem cells from the rat. Mammalian neural creststem cells and multipotent neural stem cells and their progeny can beisolated from tissues from human and nonhuman primates, equines,canines, felines, bovines, porcines, lagomorphs, etc.

The invention encompasses several important methodologicalinnovations: 1) the use of monoclonal antibodies to the low-affinityNerve Growth Factor Receptor (LNGFR) as a cell surface marker to isolateand identify neural crest stem cells, a method extensible to otherneural stem cell populations as well; 2) the development of cell culturesubstrates and medium compositions which permit the clonal expansion ofundifferentiated neural crest cells; 3) the development of culturesubstrates and medium compositions which permit the differentiation ofmammalian neural crest cells into their differentiated derivatives(including but not restricted to peripheral neurons and smooth musclecells) in clonal culture.

The invention also provides neural crest stem cells and othermultipotent neural stem cells. It is important to understand that suchcells could not be identified as stem cells without the development ofthe isolation and cell culture methodologies summarized above. Theidentification of a neural stem cell requires that several criteria bemet: 1) that the cell be an undifferentiated cell capable of generatingone or more kinds of differentiated derivatives; 2) that the cell haveextensive proliferative capacity; 3) that the cell be capable ofself-renewal or self-maintenance (Hall et al. (1989) Development106:619; Potten et al. (1990) Development 110:1001). The concept of astem cell as obligatorily capable of "unlimited" self-renewal isapplicable only to regenerating tissues such as skin or intestine. Inthe case of a developing embryo stem cells may have limited self-renewalcapacity but be stem cells nevertheless. Potten et al. (1990)Development 110:1001. The development of clonal culture methodspermitted the demonstration of criteria 1 and 2 herein. The developmentof sub-clonal culture methods (i.e., the ability to clone single neuralstem cells, and then re-clone progeny cells derived from the originalfounder cell) further permitted the demonstration herein of criterion 3.

To appreciate the significance of this demonstration, consider analternative hypothesis for cells from the neural crest: individualundifferentiated neural crest cells divide to generate both neurons andsmooth muscle cells (i.e., meet criteria 1 and 2 above), but thedaughter cells produced by these initial cell divisions are committed toproducing either neurons or smooth muscle cells, but not both. In thiscase, the neural crest cell is a progenitor cell but not a stem cell,because it does not have self-renewal capacity. If this were the case,then upon sub-cloning of neural crest cell clones, the resulting"secondary" clones could contain either neurons or smooth muscle cells,but not both. This is not observed. Rather, under certain cultureconditions, essentially all of the secondary clones contain both neuronsand smooth muscle cells, like their parent clones. This experiment thusprovides the first definitive evidence that neural progenitor cells fromany region of the nervous system have stem cell properties. In no otherset of published experiments have these stringent criteria for stem cellproperties been met, despite claims that "stem cells" have been isolatedor identified (Cattaneo et al. (1991) Trends Neurosci. 14:338; Reynoldset al. (1992) Science 255:1707) from the mammalian central nervoussystem. This in part reflects imprecise use of the term "stem cell" andin part the failure to perform adequate experimental tests to supportthe existence of such cells.

As used herein, the term "non-transformed cells" means cells which areable to grow in vitro without the need to immortalize the cells byintroduction of a virus or portions of a viral genome containing anoncogene(s) which confers altered growth properties upon cells by virtueof the expression of viral genes within the transformed cells. Theseviral genes typically have been introduced into cells by means of viralinfection or by means of transfection with DNA vectors containingisolated viral genes.

As used herein, the term "genetically-engineered cell" refers to a cellinto which a foreign (i.e., non-naturally occurring) nucleic acid, e.g.,DNA, has been introduced. The foreign nucleic acid may be introduced bya variety of techniques, including, but not limited to,calcium-phosphate-mediated transfection, DEAE-mediated transfection,microinjection, retroviral transformation, protoplast fusion andlipofection. The genetically-engineered cell may express the foreignnucleic acid in either a transient or long-term manner. In general,transient expression occurs when foreign DNA does not stably integrateinto the chromosomal DNA of the transfected cell. In contrast, long-termexpression of foreign DNA occurs when the foreign DNA has been stablyintegrated into the chromosomal DNA of the transfected cell.

As used herein, an "immortalized cell" means a cell which is capable ofgrowing indefinitely in culture due to the introduction of an"immortalizing gene(s)" which confers altered growth properties upon thecell by virtue of expression of the immortalizing gene(s) within thegenetically engineered cell. Immortalizing genes can be introduced intocells by means of viral infection or by means of transfection withvectors containing isolated viral nucleic acid encoding one or moreoncogenes. Viruses or viral oncogenes are selected which allow for theimmortalization but preferably not the transformation of cells.Immortalized cells preferably grow indefinitely in culture but do notcause tumors when introduced into animals.

As used herein, the term "transformed cell" refers to a cell having theproperties of 1) the ability to grow indefinitely in culture and 2)causing tumors upon introduction into animals. "Transformation" refersto the generation of a transformed cell.

As used herein, the term "feeder-cell independent culture" orgrammatical equivalents means the growth of cells in vitro in theabsence of a layer of different cells which generally are first platedupon a culture dish to which cells from the tissue of interest areadded. The "feeder" cells provide a substratum for the attachment of thecells from the tissue of interest and additionally serve as a source ofmitogens and survival factors. The feeder-cell independent culturesherein utilize a chemically defined substratum, for example fibronectin(FN) or poly-D-lysine (PDL) and mitogens or survival factors areprovided by supplementation of the liquid culture medium with eitherpurified factors or crude extracts from other cells or tissues.Therefore, in feeder-cell independent cultures, the cells in the culturedish are primarily cells derived from the tissue of interest and do notcontain other cell types required to support the growth of the cellsderived from the tissue of interest.

As used herein, the term "clonal density" means a density sufficientlylow enough to result in the isolation of single, non-impinging cellswhen plated in a culture dish, generally about 225 cells/100 mm culturedish.

As used herein, the term "neural crest stem cell" means a cell derivedfrom the neural crest which is characterized by having the properties(1) of selfrenewal and (2) asymmetrical division; that is, one celldivides to produce two different daughter cells with one being self(renewal) and the other being a cell having a more restricteddevelopmental potential, as compared to the parental neural crest stemcell. The foregoing, however, is not to be construed to mean that eachcell division of a neural crest stem cell gives rise to an asymmetricaldivision. It is possible that a division of a neural crest stem cell canresult only in self-renewal, in the production of more developmentallyrestricted progeny only, or in the production of a self-renewed stemcell and a cell having restricted developmental potential.

As used herein, the term "multipotent neural stem cell" refers to a cellhaving properties similar to that of a neural crest stem cell but whichis not necessarily derived from the neural crest. Rather, as describedhereinafter, such multipotent neural stem cells can be derived fromvarious other tissues including neural epithelial tissue from the brainand/or spinal cord of the adult or embryonic central nervous system orneural epithelial tissue which may be present in tissues comprising theperipheral nervous system. In addition, such multipotent neural stemcells may be derived from other tissues such as lung, bone and the likeutilizing the methods disclosed herein. It is to be understood that suchcells are not limited to multipotent cells but may comprise apluripotent cell capable of regeneration and differentiation todifferent types of neurons, glia, or smooth muscle e.g., PNS and CNSneurons, glia and smooth muscle or progenitors thereof. In this regard,it should be noted that the neural crest stem cells described herein areat least multipotent in that they are capable, under the conditionsdescribed, of self-regeneration and differentiation to some but not alltypes of neurons, glia and smooth muscle in vitro. Thus, a neural creststem cell is a multipotent neural stem cell derived from a specifictissue, i.e., the embryonic neural tube.

In most embodiments, neural crest stem cells are further characterizedby a neural cell-specific surface marker. Such surface markers inaddition to being found on neural crest stem cells may also be found onother multipotent neural stems derived therefrom, e.g., glial andneuronal progenitor cells of the peripheral nervous system (PNS) andcentral nervous system (CNS). An example is the cell surface expressionof a nerve growth factor receptor on neural crest stem cells. In rat,humans and monkeys this nerve growth factor receptor is the low-affinitynerve growth factor receptor (LNGFR). Such stem cells may also becharacterized by the expression of nestin, an intracellular intermediatefilament protein. Neural crest stem cells may be further characterizedby the absence of markers associated with mature PNS neuronal or glialcells. In rat, humans and monkeys this nerve growth factor receptor isthe low-affinity nerve growth factor receptor (LNGFR). Such stem cellsmay also be characterized by the expression of nestin, an intracellularintermediate filament protein. Neural crest stem cells may be furthercharacterized by the absence of markers associated with mature PNSneuronal or, glial cells. In the rat, such markers include sulfatide,glial fibrillary acidic protein (GFAP) and myelin protein P_(o) in PNSglial cells, and peripherin and neurofilament in PNS neuronal cells.

LNGFR is a receptor for nerve growth factor, a neurotrophic factor shownto be responsible for neuronal survival in vivo. LNGFR is found onseveral mammalian cell types including neural crest cells and Schwanncells (glial cells of the PNS) as well as on the surface of cells in theventricular zone throughout the embryonic central nervous systems. (See,e.g., Yan et al. (1988) J. Neurosci. 8:3481-3496 and Heuer, J. G et al.(1980) Neuron 5:283-296 which studied such cells in the rat and chicksystems, respectively.) Antibodies specific for LNGFR have beenidentified for LNGFR from rat monoclonal antibodies 217c (Peng, W. W. etal. (1982) Science 215:1102-1104) and 192-Ig (Brockes, J. P. et al.(1977) Nature 266:364-366 and Chandler, C. E. et al. (1984) J. Biol.Chem. 259:6882-6889) and human (Ross, A. H. et al. (1984) Proc. Natl.Acad. Sci. USA 81:6681-6685; Johnson, et al. (1986) Cell 47:545-554; Loyet al. (1990) J. Neurosci Res. 27:651-644). The monoclonal antibodyagainst human LNGFR has been reported to cross-react with LNGFR frommonkeys (Mufson, E. G. et al. (1991) J. Comp. Neurol. 308:555-575). TheDNA sequence has been determined for rat and human LNGFR (Radeke, M. J.et al. (1987) Nature 325:593-597 and Chao, M. V. et al. (1986) Science232:518-521, respectively) and is highly conserved between rat andhuman.

Using the following techniques, monoclonal antibodies specific for LNGFRfrom any desired mammalian species are generated bv first isolating thenucleic acid encoding the LNGFR protein. One protocol for obtaining suchnucleic acid sequences uses one or more nucleic acid sequences from aregion of the LNGFR gene which is highly conserved between mammalianspecies, e.g., rat and human, as a hybridization probe to screen agenomic library or a cDNA library derived from mammalian tissue from thedesired species (Sambrook, J. et al. (1989) Cold Spring HarborLaboratory Press. Molecular Cloning: A Laboratory Manual, 2nd Ed., pp.8.3-8.80, 9.47-9.58 and 11.45-11.55). The cloned LNGFR sequences arethen used to express the LNGFR protein or its extracellular (ligandbinding) domain in an expression host from which the LNGFR protein ispurified. Purification is performed using standard techniques such aschromatography on gel filtration, ion exchange or affinity resins. Thepurified LNGFR is then used to immunize an appropriate animal (e.g.,mouse, rat, rabbit, hamster) to produce polyclonal antisera and toprovide spleen cells for the generation of hybridoma cell linessecreting monoclonal antibodies specific for LNGFR of the desiredspecies (Harlow, E. et al. (1988) Cold Spring Harbor Laboratory Press,Antibodies: A Laboratory Manual, pp. 139-242).

A novel screening method can be used to detect the production ofantibody against LNGFR or any other surface marker which characterizes amultipotent neural stem cell or progeny thereof. The method can bepracticed to detect animals producing polyclonal antibodies against aparticular antigen or to identify and select hybridomas producingmonoclonal antibodies against such antigens. In this method, serum froman immunized animal or supernatent from a hybridoma culture is contactedwith a live neural cell which displays a surface marker characteristicof a particular neural cell line. Detection of whether binding hasoccurred or not is readily determined by any number of known methods Aparticularly preferred method is to use labeled antibody which isspecific for the immunoglobulins produced by the species which isimmunized with the particular antigen and which is a source forpolyclonal serum and spleen cells for hybridoma formation.

The live neural cell used in the foregoing antibody assay is dependentupon the particular surface marker for which an antibody is desired. Inthe examples, a monoclonal antibody for mouse LNGFR was identified usinga dissociated primary culture of Schwann cells. In conjunction with theassay disclosed in the examples, mouse fibroblasts acted as a negativecontrol. However, primary cultures of other cell lines can be used todetect monoclonal antibodies to LNGFR. For example, forebraincholinergic neurons or sensory neurons can be used. In addition, aprimary culture of epithelial cells can be used as a negative control.

Other markers found on neural cells include Platelet Derived GrowthFactor Receptor (PDGFR), Fibroblast Growth Factor (FGF) and Stem CellFactor Receptor (SCFR). Cells useful for detecting monoclonal antibodiesto PDGFR and FGF include primary cultures of glial cells or fibroblasts.Negative controls include cultures of epithileal cells andneuroblastomas.

SCFR is expressed on a subset of neuronal cells. Primary cultures ofmelanocytes or melanoma cells can be used to detect monoclonalantibodies to this receptor. Negative controls include primary culturesof fibroblasts and glial cells.

It is not always necessary to generate polyclonal or monoclonalantibodies that are species specific. Monoclonal antibodies against anantigenic determinant from one species may react against that antigenfrom more than one species. For example, as stated above, the antibodydirected against the human LNGFR molecule also recognizes LNGFR onmonkey cells. When cross-reactive antibodies are available, there is noneed to generate antibodies which are species specific using the methodsdescribed above.

Nestin, a second marker in the neural crest stem cell, is anintermediate filament protein primarily located intracellularly, whichhas been shown to be present in CNS neuroepithelial cells and Schwanncells in the peripheral nervous system of rats (Friedman et al. (1990)J. Comp. Neurol. 295:43-51). Monoclonal antibodies specific for ratnestin have been isolated: Rat 401, (Hockfield, S. et al. (1985) J.Neurosci. 5(12):3310-3328). A polyclonal rabbit anti-nestin antisera hasbeen reported which recognizes mouse nestin. Reynolds, D. A. et al.(1992) Science 255:1707-1710). The DNA sequences encoding the rat nestingene have been cloned. Lendahl, U. et al. (1990) Cell 60:585-595). TheseDNA sequences are used to isolate nestin clones from other mammalianspecies. These DNA sequences are then used to express the nestin proteinand monoclonal antibodies directed against various mammalian nestins aregenerated as described above for LNGFR.

Glial fibrillary acidic protein (GFAP) is an intermediate filamentprotein specifically expressed by astrocytes and glial cells of the CNSand by Schwann cells, the glial cells of the PNS (Jessen, K. R. et al.(1984) J. Neurocytology 13:923-934 and Fields, K. L. et al. (1989) J.Neuroimmuno. 8:311-330). Monoclonal antibodies specific for GFAP havebeen reported (Debus et al. (1983) Differentiation 25:193-203). Mouseand human GFAP genes have been cloned (Cowan, N. J. et al. (1985) N.Y.Acad. Sci. 455:575582 and Bongcamrudlowss, D. et al. (1991) Cancer Res.51:1553-1560, respectively). These DNA sequences are used to isolateGFAP clones from other mammalian species. These DNA sequences are thenused to express the GFAP protein and monoclonal antibodies directedagainst various mammalian GFAPs are generated as described above forLNGFR.

As used herein, the term "neuronal progenitor cell" refers to a cellwhich is intermediate between the fully differentiated neuronal cell anda precursor multipotent neural stem cell from which the fullydifferentiated neuronal cell develops. In general, such neuronalprogenitor cells are derived according to the methods described hereinfor isolating such cells from various tissues including adult andembryonic CNS and PNS tissue as well as other tissues which maypotentially contain such progenitors.

As used herein, the term "PNS neuronal progenitor cell" means a cellwhich has differentiated from a mammalian neural crest stem cell whichis committed to one or more PNS neuronal lineages and is a dividing cellbut does not yet express surface or intracellular markers found on moredifferentiated, non-dividing PNS neuronal cells. Such progenitor cellsare preferably obtained from neural crest stem cells isolated from theembryonic neural crest which have undergone further differentiation.However, equivalent cells may be derived from other tissue. When PNSneuronal progenitor cells are placed in appropriate culture conditionsthey differentiate into mature PNS neurons expressing the appropriatedifferentiation markers, for example, peripherin, neurofilament andhigh-polysialic acid neural cell adhesion molecule (high PSA-NCAM).

Peripherin, a 57 kDa intermediate filament protein, is expressed inadult rodents primarily in peripheral neurons. More limited expressionof peripherin is found in some motoneurons of the spinal cord and brainstem and a limited group of CNS neurons. Peripherin is expressed in ratembryos primarily in neurons of peripheral ganglia and in a subset ofventral and lateral motoneurons in the spinal cord. Gorham, J. D. et al.(1990) Dev. Brain Res. 57:235-248. Antibodies specific for this markerhave been identified in the rat (Portier, M. et al. (1983/84) Dev.Neurosci. 6:335-344). The DNA sequences encoding the rat peripherin genehave been cloned. Thompson, M. A. et al. (1989) Neuron 2:1043-1053.These DNA sequences are used to isolate DNA sequences for the peripheringene in other mammals that are used to express the protein and generateantibodies directed against other mammalian peripherin proteins, asdescribed above for LNGFR.

Neurofilaments are neuron-specific intermediate filament proteins. Threeneurofilament (NF) proteins have been reported: NF68, a 68 kD proteinalso called NF-L (Light); NF160, a 160 kD protein also called NF-M(Medium); NF200, a 200 kD protein also called NF-H (Heavy). In general,there is coordinate expression of all three NF proteins in neurons. TheDNA sequences encoding the rat NF200 and NF160 proteins have been cloned(Dautigny, A. et al. (1988) Biochem. Biophys. Res. Commun. 154:10991106and Napolitano, E. W. et al. (1987) J. Neurosci. 7:2590-2599,respectively). All three NF protein genes have been cloned in mice andhumans. Mouse NF68 nucleic acid sequences were reported in Lewis, S. A.et al. (1985) J. Cell Biol. 100:843-850. Mouse NF160 nucleic acidsequences were reported in Levy, E. et al. (1987) Eur. J. Biochem.166:71-77. Mouse NF200 nucleic acid sequences were reported inShneidman, P.S. et al. (1988) Mol. Brain Res. 4:217-231. In humans,nucleic acid sequences were reported for: NF68, Julien, J.-P. et al.(1987) Biochem. Biophys. Acta. 909:10-20; NF160, Myers, M. W. et al.(1987) EMBO J. 6:1617-1626; NF200, Lee, J. F. et al. (1988) EMBO J.7:1947-1955. These DNA sequences are used to produce the protein for theproduction of antibodies or to isolate other mammalian NF genes and theproteins expressed and antibodies generated for any desired species, asdescribed above for LNGFR. As used herein, the term "NF⁺ " meansexpression of one or more of the three NF proteins.

As used herein, the term "factors permissive for PNS neuronal celldifferentiation" means compounds, such as, but not limited to, proteinor steroid molecules or substrates such as FN or PDL, which permit atleast a neural crest stem cell to become restricted to the PNS neuronallineage. Such lineage-restricted progeny of neural crest stem cellsinclude PNS neuronal progenitor cells, which are at least bipotential,in that they can divide to give rise to self, as well as, more mature,non-dividing PNS neurons.

As used herein, the term "growth factors from the TGF-β superfamily"means growth factors related to transforming growth factor beta-1("TGFβ-1"). Such TGF-β superfamily growth factors may or may not exert asimilar biological effect to TGFβ-1, the prototypic member of the TGF-βsuperfamily. In recombinant TGF-β1 ("rTGF-β1") virtually all neuralcrest stem cell colonies differentiate to SM cells under specifiedculture conditions. Shah et al.(1996) Cell 85:331-343. TGFβ2 and TGFβ3yielded similar results as TGFβ1. Shah et al.(1996) Cell 85:331-343,data not shown. By way of example, members of the TGF-β superfamily ofgrowth factors include but are not limited to naturally occurringanalogues (e.g. TGFβ-2, β-3, β4),and any known synthetic or naturalanalogues of TGFβ-1 in addition to related growth factors exemplified bybone morphogenic proteins 2 and 4 ("BMP-2" and "BMP-4"). These compoundscan be purified from natural sources or may be produced by recombinantDNA techniques and may or may not be substantially pure. Variants andfragments retaining the property of causing differentiation are includedin the definition of the members of this superfamily.

As used herein, the term bone morphogenic protein ("BMP") refers to agroup of growth factors which are members of the TGF-β superfamily.Under comparable culture conditions BMP2 and BMP4 produced neurons andsome SM cells, while TGFβ1 produced only SM cells. Shah et al.(1996)Cell 85:331-343. As indicated in the examples, when neural stem cellsare contacted with certain factors permissive for neuronal and glialcell differentiation, such cells differentiated into neurons, glia and asubpopulation referred to as "O" cells. As disclosed in Example 10,these O cells are, in fact, smooth muscle cells. Thus, at least some ofthe factors which are permissive for differentiation to neuronal and/orglial cells are also permissive for the differentiation of neural stemcells to smooth muscle cells. However, as also indicated in Example 10,there are factors which are instructive for smooth muscle celldifferentiation. The growth factors described herein can be administeredindividually or in combination with each other.

In this regard, the term "instructive factor" or grammatical equivalentsrefers to one or more factors which are capable of causing thedifferentiation of neural stem cells primarily to a single lineage,e.g., glial, neuronal or smooth muscle cell. Thus, a factor which isinstructive for smooth muscle cell differentiation is one which causesdifferentiation of neural stem cells to smooth muscle cells at theexpense of the differentiation of such stem cells into other lineagessuch as glial or neuronal cells. As indicated in Example 10, mammalianserum contains one or more factors which are instructive factors for theproduction of smooth muscle cells.

Having identified that mammalian serum contains one or more instructivefactors for smooth muscle cell differentiation, such instructive factorscan be identified by fractionating mammalian serum and adding back oneor more such fractions to a neural stem cell culture to identify one ormore fractions containing instructive factors for smooth muscle celldifferentiation. Positive fractions can then be further fractionated andreassayed until the one or more components required for instructivedifferentiation to smooth muscle cells are identified.

Mammalian neural crest stem cell compositions are provided which serveas a source for neural crest cell derivatives such as neuronal and glialprogenitors of the PNS which in turn are a source of PNS neurons andglia. Methods are provided for the isolation and clonal culture ofneural crest stem cells, in the absence of feeder cells. In the examplesprovided, these methods utilize a chemically defined medium which issupplemented with chick embryo extract as a source of mitogens andsurvival factors. Factors present in the extract of chicken embryosallow the growth and self renewal of rat neural crest stem cells.However, media used to isolate and propagate rat neural crest stem cellscan be used to isolate and propagate neural crest stem cells from othermammalian species, such as human and non-human primates, equines,felines, canines, bovines, porcines, lagomorphs, etc.

Culture conditions provided herein allow the isolation self-renewal anddifferentiation of mammalian neural crest stem cells and their progeny.These culture conditions may be modified to provide a means of detectingand evaluating growth factors relevant to mammalian neural crest stemcell self-renewal and the differentiation of the stem cell and itsprogeny. These modifications include, but are not limited to, changes inthe composition of the culture medium and/or the substrate and in thespecific markers used to identify either the neural crest stem cell ortheir differentiated derivatives.

Culture conditions are provided which allow the differentiation ofmammalian neural crest stem cells into the PNS neuronal, glial andsmooth muscle lineages in the absence of feeder cell layers. In additionto liquid culture media, these culture conditions utilize a substratumcomprising fibronectin alone or in combination with poly-D-lysine. Inthe examples provided, human fibronectin is utilized for the culturingof rat neural crest stem cells and their progeny. Human fibronectin canbe used for the culturing of neural crest stem cells isolated from avianspecies as well as from any mammal, as the function of the fibronectinprotein is highly conserved among different species. Cells of manyspecies have fibronectin receptors which recognize and bind to humanfibronectin.

In order to isolate the subject neural crest stem cells, it is necessaryto separate the stem cell from other cells in the embryo. Initially,neural crest cells are obtained from mammalian embryos.

For isolation of neural crest cells from mammalian embryos, the regioncontaining the caudal-most 10 somites are dissected from early embryos(equivalent to gestational day 10.5 day in the rat). These trunksections are transferred in a balanced salt solution to chilleddepression slides, typically at 4° C., and treated with collagenase inan appropriate buffer solution such as Howard's Ringer's solution. Afterthe neural tubes are free of somites and notochords, they are platedonto fibronectin (FN)-coated culture dishes to allow the neural crestcells to migrate from the neural tube. Twenty-four hours later,following removal of the tubes with a sharpened tungsten needle, thecrest cells are removed from the FN-coated plate by treatment with aTrypsin solution, typically at 0.05%. The suspension of detached cellsis then collected by centrifugation and plated at an appropriatedensity, generally 225 cells/100 mm dish in an appropriate chemicallydefined medium. This medium is preferentially free of serum and containscomponents which permit the growth and self-renewal of neural crest stemcells. The culture dishes are coated with an appropriate substratum,typically a combination of FN and poly-D-lysine (PDL).

Procedures for the identification of neural crest stem cells includeincubating cultures of crest cells for a short period of time, generally20 minutes, at room temperature, generally about 25° C., with saturatinglevels of antibodies specific for a particular marker, e.g., LNGFR.Excess antibody is removed by rinsing the plate with an appropriatemedium, typically L15 medium (Gibco) supplemented with fresh vitamin mixand bovine serum albumin (L-15 Air). The cultures are then incubated atroom temperature with a fluorochrome labeled secondary antibody,typically Phycoerythrin R-conjugated secondary antibody (TAGO) at anappropriate dilution for about 20 minutes. Excess secondary antibodiesare then removed using an appropriate medium, such as L-15 Air. Theplates are then covered with the chemically defined growth medium andexamined with a fluorescence microscope. Individual LNGFR⁺ clones areisolated by fluorescence activated cell sorting (FACS) or, moretypically, by marking the plate under the identified clone. The markingsare typically made to a diameter of 3-4 mm, which generally allows forthe unambiguous identification of the progeny of the founder cell at anytime during an experiment. If desired, individual LNGFR⁺ clones areremoved from the original plate by trypsinization with the use ofcloning cylinders.

Procedures for permitting the differentiation of stem cells include theculturing of isolated stem cells in a medium permissive fordifferentiation to a desired lineage, such as Schwann celldifferentiation (SCD) medium. Other procedures include growth ofisolated stem cells on substrates capable of permitting differentiation,such as FN or FN and PDL.

Procedures for the serial subcloning of stem cells and their derivativesinclude the trypsinization of individual clones, as described above,followed by replating the clone on a desired substrate and culturing ina desired medium, such as a chemically defined medium suitable formaintenance of stem cells or SCD medium permissive for thedifferentiation of said neural crest stem cells. Crest cells may beidentified following serial subcloning by live-cell labeling with anantibody directed against LNGFR, as described above.

It has been demonstrated that neurogulin/GGF can instructively influencemultipotent, self-renewing rodent neural crest stem cells(NCSCs)(Stemple and Anderson (1992) Cell 71:973-985) to differentiate to gliain vitro (Shah et al. (1994) Cell 77:349-360; while this studydemonstrated that the fate could be promoted by an environmental signal,it left open the question of how alternative fates might be chosen. Forexample, the neuronal fate of NCSCs, might be promoted by otherextrinsic cues. Alternatively, neural crest cells might be predisposedto select a neuronal fate in the absence of extrinsic influence. Indeed,in many systems developmental decision have been suggested to involveone fate that is promoted by an extracellular signal and an alternativefate that is assumed in the absence of that signal, as if by default.(Raff (1989) Science 243:1450-1455; Kelly and Melton (1995) TrendsGenet. 11:273-278; Shah et al.(1996) Cell 85:331-343.)

The mechanisms controlling binary fate decisions by developmentallyequivalent cells are being rapidly elucidated by genetic analysis(Greenwald and Rubin (1992) Cell 68:271-281; Ghysen et al.(1993) GenesDev. 7:723733. In contrast, the process whereby a multiplicity ofdifferentiated cell types is generated from pluripotent stem cells isless well understood. This problem has been studied in the context ofhematopoiesis, in vertebrates (for reviews, see Dexter et al.(1990)Phil. Trans. R. Soc. Lond. (B) 327:85-98; Shah et al.(1996) Cell85:331-343). In this system, growth factors such as erythropoietin (EPO)have been isolated that influence the development of cells in aparticular lineage (Clark and Kaman (1987) Science 236:1229-1237;Metcalf (1989) Nature 339:27-30; Krantz (1991) Blood 77:419-434. A majorissue has been whether these growth factors act by instructingmultipotent cells to commit to one lineage at the expense of others(Metcalf and Burgess (1982) Cell. Physiol. 111:27 5-283, or whether theyprevent the death of lineage-committed progenitor that have beengenerated by a stochastic mechanism (see Ogawa (1993) Blood81:2844-2853, and references therein). The available evidence favors thelatter alternative. Shah et al.(1996) Cell 85:331-343.

The methods described herein provide the basis of functional assayswhich allow for the identification and production of cellularcompositions of mammalian cells which have properties characteristic ofneural crest stem cells, glial, neuronal, smooth muscle progenitor cellsor multipotent stem cell precursor of such progenitor cells. In order toisolate such cells from tissues other than embryonic neural tubes, it isnecessary to separate the progenitor and/or multipotent stem cells fromother cells in the tissue. The methods presented in the examples for theisolation of neural crest stem cells from neural tubes can be readilyadapted for other tissues by one skilled in the art. First, a singlecell suspension is made from the tissue; the method used to make thissuspension will vary depending on the tissue utilized. For example, sometissues require mechanical disruption of the tissue while other tissuesrequire digestion with proteolytic enzymes alone or in combination withmechanical disruption in order to create the single cell suspension.Tissues such as blood already exists as a single cell suspension and nofurther treatment is required to generate a suspension, althoughhypotonic lysis of red blood cells may be desirable. Once the singlecell suspension is generated it may be enriched for cells expressingLNGFR or other neural cell-specific markers on their surface. Oneprotocol for the enrichment for LNGFR⁺ cells is by incubating the cellsuspension with antibodies specific for LNGFR and isolating the LNGFR⁺cells. Enrichment for cells expressing a neural cell-specific surfacemarker is particularly desirable when these cells represent a smallpercentage (less than 5%) of the starting population. The isolation ofcells which have complexed with an antibody for a neural cell-specificsurface marker such as is carried out using any physical method forisolating antibody-labeled cells. Such methods includefluorescent-activated cell sorting in which case the cells, in general,are further labeled with a fluorescent secondary antibody that binds theanti-LNGFR antibody, e.g., mouse anti-LNGFR and fluorescein label goatanti-mouse IgG; panning in which case the antibody-labeled cells areincubated on a tissue-culture plate coated with a secondary antibody;Avidin-sepharose chromatography in which the anti-LNGFR antibody isbiotinylated prior to incubation with the cell suspension so that thecomplexed cells can be recovered on an affinity matrix containing avidin(i.e., where the antibody is an antibody conjugate with one of themembers of a binding pair); or by use of magnetic beads coated with anappropriate anti-antibody so that the labeled LNGFR-expressing cells canbe separated from the unlabeled cells with the use of a magnet. All ofthe foregoing cell isolation procedures are standard publishedprocedures that have been used previously with other antibodies andother cells.

The use of antibodies specific for neural stem cell-specific surfacemarkers results in the isolation of multipotent neural stem cells fromtissues other than embryonic neural tubes. For example, as previouslyindicated, LNGFR is expressed in cells of the ventricular zonethroughout the embryonic central nervous system of the rat and chick.This implies that other mammalian species have a similar pattern ofLNGFR expression and studies in human with monoclonal antibodies againstthe human LNGFR (Loy, et al. (1990) J. Neurosci. Res. 27:651-654) areconsistent with this expectation. Since cells from the ventricular zone(Cattaneo et al. (1991) Trends Neurosci. 14:338-340; Reynolds et al.(1992) Science 255:1707-1710) are likely to be stem cells (Hall et al.(1989) Development 106:619-633; Potter et al. (1990) Development110:1001-1020) antibodies to neural cell-specific surface markers shouldprove useful in isolating multipotent neural stem cells from the centraland peripheral nervous systems and from other tissue sources.

Alternatively, or in conjunction with the above immuno-isolation step,the cells are plated at clonal density, generally 225 cells/100 mm dish,in an appropriate chemically defined medium on a suitable substrate asdescribed in the examples for isolation of rat neural crest stem cells.The presence of neural crest-like stem cells (e.g., a multipotent neuralstem cell) is confirmed by demonstrating that a single cell can bothself-renew and differentiate to members of at least the PNS neuronal,glial and smooth muscle lineages utilizing the culture conditionsdescribed herein. Other types of multipotent neural stem cells areidentified by differentiation to other cell types such as CNS neural orglial cells or their progenitors. Depending upon the source of thetissue used in the foregoing methods, multipotent neural stem cells maynot be obtained. Rather, further differentiated cell types such asglial, neuronal or smooth muscle progenitor cells may be obtained.

The identification of the large, flat cells that develop in the presenceof TGFβ1 (and, to a lesser extent, in rBMP2) as SM cells is based ontheir morphology (Chamley-Campbell et al. (1979) Physiol. Rev. 59:1-61and expression of two SM-specific markers, calponin and (αSMA (Skalli etal.(1986) J. Cell Biol. 103:2787-2796; Gimona et al., (1990) FEBS Lett.274:159-162). Although each of these markers can occasionally beexpressed by some non-SM cells, the co-expression of both markers bymany individual cells in cultures makes it likely that these cells arein fact SM. SM cells are one of the normal derivatives of the neuralcrest, although in avians they derive from an anterior region of theneural crest (the cardiac neural crest; Kirby (1987) Pediatr. Res.21:219-224), rather than from the trunk region (which corresponds to theregion from which our NCSCs are obtained). The trunk crest has thecapacity to give rise to SM if transplanted to anterior regions(Nakamura and Ayer-Le Lievre (1982) J. Embryol. Exp. Morphol. 70:1-19.Therefore, the ability to elicit SM differentiation from rodent trunkNCSCs may reflect a developmental potential that is available to thesecells in vivo. The available fate mapping data (Serbedzija et al.(1990)Development 108:605-612) do not exclude a contribution of trunk neuralcrest to SM in mammals.

Although the development of SM cells is of considerable relevance forhuman disease (Kirby and Waldo (1990) Circulation 82:332-340, theirdevelopment from precursor cells in mammals is poorly understood (seeSchwartz et al. (1990; Owens, (1995) Physiol. Rev. 75:487-517 andreferences therein). While SM cell differentiation has been obtainedfrom cell lines such as ES-like cells (Edwards et al.(1983) Mol. Cell.Biol. 3:2280-2286, the present invention represents the first case inwhich de novo differentiation of these cells from a naturally occurringprecursor has been elicited in vitro. Such a system should open the wayto further studies aimed at understanding the factors that control thedifferentiation and maturation of this important cell type.Chamley-Campbell et al. (1979) Physiol. Rev. 59:1-61.

TGFβ1 super family members may be utilized to instructively influencecell fate decisions, rather than selectively support survival oflineage-committed progenitors. Shah et al.(1996) Cell 85:331-343.Members of the TGF-β superfamily of growth factors are expressed atsites where autonomic neurons differentiate. Shah et al.(1996) Cell85:331-343. For example, bone morphogenic protein 2 (BMP2) promotesrapid induction of the autonomic lineage-specific basic-helix-loop-helixprotein MASH 1 and autonomic neurogenesis in vitro. Some SM celldifferentiation is also observed in BMP2 in contrast to TGFβ1, theprototypic member of the TGFβ superfamily, which drives virtually allNCSCs to a SM fate. Shah et al.(1996) Cell 85:331-343.

A clonal culture system has been developed(Stemple and Anderson (1992)Cell 71:973-985) which has permitted detailed investigation of theaction of growth factors on rodent neural crest cells. Initially, thepromotion of neuregulin/GGF on glial as opposed to neuronaldifferentiation was demonstrated. Shah et al. (1994) Cell 77:349-360.More recently, SM differentiation has been added to the NCSC repertoireand as being promoted by TGFβ1. In contrast, a related factor, BMP2/4,promotes primarily autonomic neuronal differentiation although some SMdifferentiation is observed. Clonal analysis and serial observations ofliving clones strongly indicates that both TGFβ1 and BMP2 actinstructively rather than selectively. The expression patterns of BMP2/4(Bitgood and McMahon (1995) Dev. Biol. 172:126-138; Lyons et al. (1995)Mech. Dev. 50:71-83, TGFβ1 (Akhurst et al.(1990) Development108:645-656; Millan et al. (1991) Development 111:131-144 ; Dickson etal. (1993) Development 117:625-639 and neurogulin (Marchionni et al.(1993) Nature 362:312-318; Meyer and Burchmeier (1994) Proc. Natl. Sci.USA 91:1064-1068; Shah et al. (1994) Cell 77:349-360) in vivo areconsistent with the roles suggested for them by these in vitroexperiments.

Transplantation assay systems described herein provide the basis offunctional assays which allow for the identification of mammalian cellswhich have properties characteristic of neural crest stem cells,multipotent neural stem cells and/or neuronal, glial or smooth muscleprogenitor cells. Cells of interest, identified by either the in vivo orin vitro assays described above, are transplanted into mammalian hostsusing standard surgical procedures. Using standard techniques, it ispossible to deliver neural crest cells to a developing mammalian oravian embryo or to any tissue or compartment of the adult animal (e.g.,brain, peritoneal cavity, etc.). The transplanted cells and theirprogeny are distinguished from the host cells by the presence of speciesspecific antigens or by the expression of an introduced marker gene. Thetransplanted cells and their progeny are also stained for markers ofmature neurons and glia in order to examine the developmental potentialof the transplanted cells. This transplantation assay provides a meansto identify neural crest stem cells by their functional properties inaddition to the in vitro culture assays described above.

Additionally, the transplantation of cells having characteristics ofmultipotent neural stem cells, neural crest stem cells or progenitors ofneuronal, glial or smooth muscle cells provides a means to investigatethe therapeutic potential of these cells for neurological disorders ofthe PNS and CNS in animal models. Examples of PNS disorders in miceinclude the trembler and shiverer strains. The trembler mutationinvolves a defect in the structural gene for peripheral myelin protein22 (PMP22). This mutation results in the defective myelination of axonsin the PNS. An analogous disorder is seen in humans, Charcot-Marie-Toothsyndrome, which results in progressive neuropathic muscular atrophy.

The shiverer mutation in mice results in a severe myelin deficiencythroughout the CNS and a moderate hypo-myelination in the PNS. Severeshivering episodes are seen 12 days after birth. An analogous disorderis seen in humans, Guillaum-Barre' disease, which is characterized by anacute febrile polyneuritis.

Cells having characteristics of multipotent neural stem cells, neuralcrest stem cells or neuronal, glial or smooth muscle progenitors of thePNS or CNS (identified by either in vitro or in vivo assays) areintroduced into a mammal exhibiting a neurological disorder to examinethe therapeutic potential of these cells. These cells are preferablyisolated from a mammal having similar MHC genotypes or the host mammalis immunosuppressed using drugs such as cyclosporin A. The cells areinjected into an area containing various peripheral nerves known to beeffected in a particular mammal or into the spinal cord or brain formammals which show involvement of the CNS. The cells are injected at arange of concentrations to determine the optimal concentration into thedesired site. Alternatively, the cells are introduced in a plasma clotor collagen gel to prevent rapid dispersal of cells from the site ofinjection. The effect of this treatment on the neurological status ofthe model animal is noted. Desired therapeutic effects in the abovemutant mice include the reduction or cessation of seizures or improvedmovement of lower motor extremities.

There is strong interest in identifying the multipotent neural stemcells such as the neural crest stem cell and defining culture conditionswhich allow the clonal propagation and differentiation of said stemcells. Having possession of a multipotent neural stem cell or a neuralcrest stem cell allows for identification of growth factors associatedwith self regeneration. In addition, there may be as yet undiscoveredgrowth factors associated with (1) with the early steps of restrictionof the stem cell to a particular lineage; (2) the prevention of suchrestriction; and (3) the negative control of the proliferation of thestem cell or its derivatives. The multipotent neural stem cell, neuralcrest stem cell, progeny thereof or immortalized cell lines derivedtherefrom are useful to: (1) detect and evaluate growth factors relevantto stem cell regeneration; (2) detect and isolate ligands, such asgrowth factors or drugs, which bind to receptors expressed on thesurface of such cells or their differentiated progeny (e.g., GlialGrowth Factor (GGF), Heregulin and Neu Differentiation Factor (NDF));(3) provide a source of cells which express or secrete growth factorsspecific to multipotent neural stem cells; (4) detect and evaluate othergrowth factors relevant to differentiation of stem cell derivatives,such as neurons, glia and smooth muscle; (5) produce various neural stemcell derivatives, including both the progenitors and mature cells of agiven lineage and (6) provide a source of cells useful for treatingneurological diseases of the PNS and CNS in model animal systems and inhumans. The culture conditions used herein allow for the growth anddifferentiation of stem cells in vitro and provide a functional assaywhereby mammalian tissues can be assayed for the presence of cellshaving the characteristics of neural stem cells. The transplantationassay described herein also provides a functional assay wherebymammalian neural stem cells may be identified.

Once identified and propagated, the stem cells may be subsequentlypreferentially differentiated into neurons or smooth muscle cells usingmedia containing growth factors. The use of growth factors from theTGF-β superfamily is illustrated in Example 11, as exemplified by TGF-βand BMP2. The appropriate dose of TGF-p superfamily growth factors maybe determined in a number of ways which will depend on the TGF-βsuperfamily growth factor used. As shown for TGF-β and BMP2 in FIG. 24,an initial range of doses may be tested and an appropriate range set.For example, 0.0001 to about 100 nM may be used with a preferred rangefrom about 0.001 to about 10 nM. The most preferred dose range may varydepending upon the TGF-β superfamily growth factor used. Shah etal.(1996) Cell 85:331-343.

It is understood by those skilled in the art that this dose may varydepending on the purity and particular preparation of the TGF-βsuperfamily growth factor. In the most preferred embodiment, the TGF-βsuperfamily growth factor is substantially pure. In other embodiments,the TGF-β superfamily growth factor is in culture media and may be oneof many ingredients in the culture media.

While the examples have focused on stem cells derived from theperipheral nervous system (PNS), i.e. from the neural crest, thoseskilled in the art would appreciate that the use of TGF-β superfamilygrowth factors to differentiate stem cells into neurons and smoothmuscle cells is not limited to PNS-derived cells. Rather, the method canbe used on stem cells derived from the central nervous system (CNS).Putative CNS stem cells have been reported (see WO 93/01275 and Reynoldet al., Science 255:1707 (1992)). The effective dosages for CNS stemcells can be determined in the same way as for PNS-derived stem cells.

As indicated in the examples, neural crest stem cells have been passagedfor at least six-ten generations in culture. Although it may beunnecessary to immortalize those or other multipotent neural stem celllines or progenitor cell lines obtained by the methods described herein,once a cell line has been obtained it may be immortalized to yield acontinuously growing cell line useful for screening trophic ordifferentiation factors or for developing experimental transplantationtherapies in animals. Such immortalization can be obtained inmultipotent neural stem cells or progenitors of glial, neuronal andsmooth muscle cells by genetic modification of such cells to introducean immortalizing gene.

Examples of immortalizing genes include: (1) nuclear oncogenes such asv-myc, N-myc, T antigen and Ewing's sarcoma oncogene (Fredericksen etal. (1988) Neuron 1:439-448; Bartlett, P. et al. (1988) Proc. Natl.Acad. Sci. USA 85:3255-3259, and Snyder, E. Y. et al. (1992) Cell68:33-51), (2) cytoplasmic oncogenes such as bcr-abl and neurofibromin(Solomon, E. et al. (1991) Science 254:1153-1160), (3) membraneoncogenes such as neu and ret (Aaronson, A.S.A (1991) Science254:1153-1161), (4) tumor suppressor genes such as mutant p53 and mutantRb (retinoblastoma) (Weinberg, R. A. (1991) Science 254:1138-1146), and(5) other immortalizing genes such as Notch dominant negative (Coffinan,C. R. et al. (1993) Cell 23:659-671). Particularly preferred oncogenesinclude v-myc and the SV40 T antigen.

Foreign (heterologous) nucleic acid may be introduced or transfectedinto multipotent neural stem cells or their progeny. A multipotentneural stem cell or its progeny which harbors foreign DNA is said to bea genetically-engineered cell. The foreign DNA may be introduced using avariety of techniques. In a preferred embodiment, foreign DNA isintroduced into multipotent neural stem cells using the technique ofretroviral transfection. Recombinant retroviruses harboring the gene(s)of interest are used to introduce marker genes, such as the E. coliβ-galactosidase (lacZ) gene, or oncogenes. The recombinant retrovirusesare produced in packaging cell lines to produce culture supernatantshaving a high titer of virus particles (generally 10⁵ to 10⁶ pfu/ml).The recombinant viral particles are used to infect cultures of theneural stem cells or their progeny by incubating the cell cultures withmedium containing the viral particles and 8 μg/ml polybrene for threehours. Following retroviral infection, the cells are rinsed and culturedin standard medium. The infected cells are then analyzed for the uptakeand expression of the foreign DNA. The cells may be subjected toselective conditions which select for cells that have taken up andexpressed a selectable marker gene.

In another preferred embodiment, the foreign DNA is introduced using thetechnique of calcium-phosphate-mediated transfection. Acalcium-phosphate precipitate containing DNA encoding the gene(s) ofinterest is prepared using the technique of Wigler et al. (1979) Proc.Natl. Acad. Sci. USA 76:1373-1376. Cultures of the neural stem cells ortheir progeny are established in tissue culture dishes. Twenty fourhours after plating the cells, the calcium phosphate precipitatecontaining approximately 20 μg/ml of the foreign DNA is added. The cellsare incubated at room temperature for 20 minutes. Tissue culture mediumcontaining 30 μM chloroquine is added and the cells are incubatedovernight at 37° C. Following transfection, the cells are analyzed forthe uptake and expression of the foreign DNA. The cells may be subjectedto selection conditions which select for cells that have taken up andexpressed a selectable marker gene.

The following is presented by way of example and is not to be construedas a limitation on the scope of the invention. Further, all referencesreferred to herein are expressly incorporated by reference.

EXAMPLE 1 Preparation of Neural Crest Cells

For a given preparation 5-10 timed pregnant female Sprague-Dawley rats(Simonson Laboratories, Gilroy, Calif.) were killed by CO₂ asphyxiation.Embryos were removed and placed into Hank's Balanced Salt Solution(HBSS) (Gibco, Grand Island, N.Y.) at 4° C. for 2-4 hours. Under adissecting microscope, at room temperature, a block of tissue from aregion corresponding to approximately the caudal most 10 somites wasdissected from each embryo using an L-shaped electrolytically sharpenedtungsten needle. Trunk sections were transferred in HBSS into one wellof a 3 well depression slide that had been chilled to 4° C. Trunksections were treated with collagenase (152 units/mg) (WorthingtonBiochemical, Freehold, N.J.) made to a concentration of 0.75 mg/ml inHoward's Ringer's solution (per 1 liter of dH₂ O: NaCl 7.2g; CaCl₂0.17g; KCl 0.37g) and sterilized, by passage through a 0.22 μm filterprior to use. The collagenase solution was exchanged at least 3 timesand with each exchange the trunk sections were vigorously triturated bypassage through a pasteur pipet. After incubation at 37° C. for 20minutes in humidified CO₂ atmosphere, the trunk sections were trituratedvery gently until most of the neural tubes were free and clean ofsomites and notochords. The collagenase solution was quenched byrepeated exchanges with cold complete medium (described below). Theneural tubes were plated onto fibronectin-coated (substrate preparationis described below) 60 mm tissue culture dishes (Corning, Corning, N.Y.)that had been rinsed with complete medium. After a 30 minute incubationto allow the neural tubes to attach, dishes were flooded with 5 ml ofmedium. After a 24 hour culture period, using an L-shapedelectrolytically sharpened tungsten needle and an inverted phasecontrast microscope equipped with a 4× objective lens, each neural tubewas carefully scraped away from the neural crest cells that had migratedonto the substrate. Crest cells were removed by a 2 minute 37° C.treatment with 0.05% Trypsin solution (Gibco). The cells werecentrifuged for 4 minutes at 2000 r.p.m. and the pellet was resuspendedinto 1 ml of fresh complete medium. Typically the cells were plated at adensity of 225 cells/100 mm dish.

Substrate Preparation

A. Fibronectin (FN) Substrate

Tissue culture dishes were coated with human plasma fibronectin (NewYork Blood Center, New York, N.Y.) in the following way. Lyophilizedfibronectin was resuspended in sterile distilled water (dH₂ O) to aconcentration of 10 mg/ml and stored at -80° C. until used. Thefibronectin stock was diluted to a concentration of 250 mg/ml inDulbecco's phosphate buffered saline (D-PBS) (Gibco). The fibronectinsolution was then applied to tissue culture dishes and immediatelywithdrawn.

B. Poly-D-Lysine (PDL) and FN Substrate

Sterile poly-D-Lysine (PDL) was dissolved in dH₂ O to as concentrationof 0.5 mg/ml. The PDL solution was applied to tissue culture plates andimmediately withdrawn. The plates were allowed to dry at roomtemperature, rinsed with 5 ml of dH₂ O and allowed to dry again.Fibronectin was then applied, as described above, over the PDL.

EXAMPLE 2 Development of a Defined Medium for the Growth of Rat NeuralCrest Stem Cells

A serum-free, chemically defined basal medium was developed based on theformulations of several existing defined media. This basal mediumconsists of L15-CO₂ formulated as described by Hawrot, E. et al. (1979)Methods in Enzymology 58:574-583 supplemented with additives describedby Bottenstein, J. E. et al. (1979) Proc. Natl. Acad. Sci. USA76:514-517 and further supplemented with the additives described bySieber-Blum, M. et al. (1985) Exp. Cell Res. 158:267-272. The finalrecipe is given here: to L15-CO₂ add, 100 μg/ml transferrin (Calbiochem,San Diego, Calif.), 5 μg/ml insulin (Sigma, St. Louis, Mo.), 16 μg/mlputrescine (Sigma), 20 nM progesterone (Sigma), 30 nM selenious acid(Sigma), 1 mg/ml bovine serum albumin, crystallized (Gibco), 39 pg/mldexamethasone (Sigma), 35 ng/ml retinoic acid (Sigma), 5 μg/ml α-d,1-tocopherol (Sigma), 63 μg/ml β-hydroxybuyrate (Sigma), 25 ng/ml cobaltchloride (Sigma), 1 μg/ml biotin (Sigma), 10 ng/ml oleic acid (Sigma),3.6 mg/ml glycerol, 100 ng/ml α-melanocyte stimulating hormone (Sigma),10 ng/ml prostaglandin El (Sigma), 67.5 ng/ml triiodothyronine (AldrichChemical Company, Milwaukee, Wis.), 100 ng/ml epidermal growth factor(Upstate Biotechnology, Inc., Lake Placid, N.Y.), 4 ng/ml bFGF (UBI),and 20 ng/ml 2.55 NGF (UBI).

To allow the growth and regeneration of neural crest stem cells infeeder cell-independent cultures, it was necessary to supplement thebasal medium with 10% chick embryo extract (CEE). This supplementedmedium is termed complete medium.

CEE is prepared as follows: chicken eggs were incubated for 11 days at38° C. in a humidified atmosphere. Eggs were washed and the embryos wereremoved, and placed into a petri dish containing sterile MinimalEssential Medium (MEM with Glutamine and Earle's salts) (Gibco) at 4° C.Approximately 10 embryos each were macerated by passage through a 30 mlsyringe into a 50 ml test tube (Corning). This typically produced 25 mlof volume. To each 25 ml was added 25 ml of MEM. The tubes were rockedat 4° C. for 1 hour. Sterile hyaluronidase (1 mg/25 g of embryo) (Sigma)was added and the mixture was centrifuged for 6 hours at 30,000 g. Thesupernatant was collected, passed first through a 0.45 μm filter, thenthrough a 0.22 μm filter and stored at -80° C. until used.

At the low cell densities necessary for survival and proliferation ofindividual neural crest cells, either fetal calf serum (FCS, JRScientific) or CEE was required, in addition to the basal medium, forclone formation. When FCS was used to supplement the medium, it was heatinactivated by treatment at 55° C. for 30 minutes. FCS was stored at-20° C. and passed through a 0.22 μm filter prior to use.

CEE is preferred as a supplement, as in the presence of FCS, most of thecells derived from the neural crest exhibit a flattened, fibroblasticmorphology and expression of LNGFR is extinguished. In the absence ofboth FCS and CEE, clone formation from neural crest cells was greatlyattenuated.

EXAMPLE 3 Isolation and Cloning of Multipotent Rat Neural Crest Cells

A. Identification of Antibody Markers Expressed by Neural Crest Cells

In order to identify and isolate rat neural crest cells, it wasnecessary to identify antibody markers that could be used to recognizethese cells. When E10.5 neural tubes were explanted onto a fibronectin(FN) substratum, many of the neural crest cells that emigrated from theneural tubes over the next 24 hours expressed the low-affinity NGFreceptor (LNGFR), recognized by monoclonal antibodies 192-Ig and 217c.The outgrowth of neural crest cells from the dorsal side of theexplanted neural tube following 24 hours growth in culture is shown inFIG. 1, panel A. FIG. 1, panel B shows the expression of LNGFR (greenflorescence) and nest in (red fluorescence) in neural crest cells.

Neural crest cells were labeled with antibodies as follows: For cellsurface antigens, such as LNGFR, it was possible to label the livingcells in culture.

The cultures were incubated with primary antibody solution for 20minutes at room temperature. The cultures were washed twice with L15medium (Gibco) supplemented with 1:1:2, fresh vitamin mix (FVM) (Hawrot,E. et al. (1979), ibid), and 1 mg/ml bovine serum albumin (L15 Air). Thecultures were then incubated for 20 minutes at room temperature withPhycoerythrin R conjugated secondary antibody (TAGO) at a dilution of1:200 in L-15 Air. The cultures were then rinsed twice with L-15 Air andplaced back in their original medium and examined with a fluorescencemicroscope. Rabbit anti-LNGFR antiserum (Weskamp, G. et al. (1991)Neuron 6:649-663) was a kind gift of Gisela Weskamp, University ofCalifornia, San Francisco and was used at a 1:1000 dilution. Monoclonalanti-NCAM antibody 5A5 (Dodd, J. et al. (1988) Neuron 1:105-116) andmonoclonal anti-sulfatide antibody O₄ (Sommer, I. et al. (1981) Dev.Biol. 83:311-327) were obtained as hybridoma cells from theDevelopmental Studies Hybridoma Bank (Johns Hopkins University,Baltimore, Md.) and prepared as described by the provider.

In order to label cells with antibodies directed against intracellularproteins, it was necessary to fix and permeabilize the cells prior tolabeling. For most of the immunocytochemistry, formaldehyde fixation wasdone. Formaldehyde solution 37% was diluted 1:10 into S-MEM with 1 mMHEPES buffer (Gibco). Culture were treated for 10 minutes at roomtemperature with the 3.7% formaldehyde solution and then rinsed 3 timeswith D-PBS (Gibco).

For some intermediate filament proteins (NF and GFAP) formaldehydefixation was not possible. Cultures were fixed by treatment with asolution of 95% ethanol and 5% glacial acetic acid at -20° C. for 20minutes.

For the staining of cytoplasmic antigens, fixed cells were first treatedwith a blocking solution comprising D-PBS, 0. 1% Tween-20 (Bio-RadLaboratories, Richmond, Calif.) and 10% heat inactivated normal goatserum (NGS) for 15 minutes at room temperature. Primary antibodies werediluted with a solution of D-PBS, 0. 1% Tween-20 and 5% NGS. The fixedcells were incubated overnight at 4° C. in primary antibody solutionthen rinsed twice with DPBS, 0.05% Tween-20. Fluorescent secondaryantibodies were diluted with D-PBS, 1% NGS and applied to cells for 1hour at room temperature. The cells were rinsed twice with D-PBS, 0.05%Tween-20. To prevent photobleaching, a solution of 8 mg/ml N-propylgallate in glycerol was placed over the stained cells prior tofluorescence microscopy.

Mouse monoclonal anti-GFAP, G-A-5 (Debus et al. (1983) Differentiation25:193-203) was purchased from Sigma and used at a 1:100 dilution. Mousemonoclonal anti-NF200, SMI39 was purchased from Sternberger MonoclonalsInc., Baltimore, Md. and used at a 1:100 dilution. SM139 reactivity isequivalent to the 06-53 monoclonal antibody described by Sternberger,L.A. et al. (1983) Proc. Natl. Acad. Sci. USA 80:6126-6130. Purifiedrabbit antibodies to peripherin (preparation 199-6) was obtained fromDr. Linda Parysek, University of Cincinnati, Ohio and was used at adilution of 1:500.

Flow-cytometric analysis indicated that greater than 70% of the neuralcrest cells show some LNGFR immunoreactivity (FIG. 1, panel D).Approximately 25% of the neural crest cells expressed high levels ofLNGFR. In some experiments, neural crest cells expressing high levels ofLNGFR were further purified by labeling with 192-Ig (anti-LNGFR) andfluorescence-activated cell sorting (FACS). For single cell analysis,however, it proved more convenient to plate the bulk neural crest cellpopulation at clonal density, and then subsequently identifyLNGFR-positive cells by live cell-labeling with 192-Ig.

Most or all of the neural crest cells also expressed nestin, anintermediate filament protein found in CNS neuroepithelial cells. Anindividual neural crest cell co-expressing both nestin and LNGFR isshown in FIG. 2, panels A-C. Panel A shows the individual neural crestcell in phase contrast.

Panels B and C show this cell following staining with both anti-LNGFR(panel B) and anti-nestin (panel C). FIG. 2, panels D-F show that theclonal progeny of this nestin⁺, LNGFR⁺ neural crest cell also co-expressnestin and LNGFR.

B. Cloning of Multipotent Neural Crest Cells

To define the developmental potential of individual neural crest cells,conditions were established that permit the growth of these cells inclonal culture. FIG. 3 provides a flow chart depicting the followingcell cloning experiments. In FIG. 3, plating medium refers to thecomplete medium, described above and differentiation medium refers toSCD medium, described below. Using an FCS-free, CEE-containing medium(complete or plating medium), single neural crest cells (FIG. 4, panelA, phase contrast and panel B, LNGFR staining) were plated on a FN/PDLsubstratum and allowed to proliferate and differentiate. After 9-14days, many of the clones founded by single neural crest cells were largeand contained cells with a neuronal morphology (FIG. 4, panel C, phasecontrast). Quantification indicated that >60% of the clones contained amixture of neuronal and non-neuronal cells (see below). These neuronalcells could be labeled by antibodies to panneuronal markers such asneurofilament (FIG. 4, panel E, anti-NF160 staining) andhigh-polysialyic acid (PSA) NCAM (FIG. 4, panel D, anti-NCAM staining),as well as by an antibody to peripherin, an intermediate filamentprotein that is preferentially expressed by peripheral nervous system(PNS) neurons (FIG. 4, panel F). Importantly, these neurons did notexpress either nestin or LNGFR, indicating that they have lost the twomarkers that characterize the undifferentiated neural crest cell. Theneuron-containing clones also contained non-neuronal cells. These cellscontinued to express LNGFR and nestin, in contrast to the neurons, anddisplayed an elongated morphology characteristic of Schwann cells. Whileimmature Schwann cells are known to express both LNGFR and nestin, thesemarkers are insufficient to identify Schwann cells in this system sincethey are expressed by the neural crest precursor cell as well.Expression of more definitive Schwann cell markers was elicited bytransferring the cells into a medium known to enhance Schwann celldifferentiation. This medium, called Schwann cell differentiation (SCD)medium, contained both 10% FCS and 5 μM forskolin, an activator ofadenylate cyclase.

FIG. 5 shows the expression of a Schwann cell phenotype by neuralcrestderived glia. Clones plated initially on FN were allowed to growfor a week in complete medium, then transferred into SCD medium andallowed to grow for another 1-2 weeks prior to fixation andimmunocytochemistry. Cells of two morphologies, one elongated and theother flattened can be seen in phase contrast (Panels A and D). Todemonstrate concordant expression of three markers, LNGFR, O₄ and GFAP,two different double-labeling experiments were performed. Living cellswere surface-labeled with monoclonal antiLNGFR 192IgG (Panel B) andmonoclonal O₄ IgM (Panel C) and postfixed. In parallel, other cells fromthe same clone were first surface-labeled with O₄ and then fixed withacid-ethanol, permeabilized and stained with anti-GFAP (IgG). Note thatLNGFR⁺ cells (Panel B) are O₄ ⁺ and that most or all of the O₄ ⁺ cellsare also GFAP⁺ (Panels E and F). The quality of the O₄ staining in(Panel E) appears different from that in (Panel C) because aredistribution of the antigen occurs following acid-ethanol fixation. InPanel C, the flattened O₄ ⁺ cells are more weakly stained for LNGFR(Panel B). Such flattening is indicative of myelination, and isconsistent with the fact that Schwann cells undergoing myelinationdown-regulate LNGFR and up-regulate O₄.

Following 5-10 days in SCD medium, most or all of the non-neuronal cellsin the clones expressed glial fibrillary acidic protein (GFAP), anintermediate filament specific to glial cells, and sulfatide, acell-surface glycolipid recognized by the monoclonal antibody O₄.Triple-labeling of such "mature" clones with polyclonal anti-peripherinand monoclonal O₄ and anti-GFAP antibodies revealed that sulfatide andGFAP were not expressed by the peripherin-positive neurons and thatthese two glial markers were coincident in the non-neuronal cellpopulation (FIG. 6). FIG. 6 shows a clone from a single founder cell inphase contrast (Panel A) which expresses LNGFR (Panel B). This clone wasallowed to proliferate and differentiate in complete medium (containingCEE and lacking serum) and then transferred into SCD medium (containingserum and forskol in). After approximately 10 days, the culture wasfixed and triple-labeled with rabbit anti-peripherin (Panels C and D, ingreen/yellow), anti-GFAP (IgG) (Panel C, in red) and O₄ (IgM) (Panel D,blue). Panels C and D are two separate fields from the same clone.

Although GFAP is expressed by astrocytes and sulfatide is expressed byoligodendrocytes in the CNS, the co-expression of these two markers inthe same cell is unique to peripheral glial cells (Jessen, K. R. et al.(1990) Devel. 109:91-103 and Mirsky, R. et al. (1990) Devel.109:105-116).

Therefore, these data indicate that single neural crest cells expressingnestin and LNGFR are able to give rise to clones of differentiated cellscontaining both peripheral neurons and glia. Differentiation to theneuronaoenotype involves both the loss of LNGFR and nestin expression,and the gain of neuronal markers such as neurofilament, high PSA-NCAMand peripherin. On the other hand, in the glial lineage LNGFR and nestinexpression persist, and additional glial markers (GFAP and O₄) areacquired. All clones that produced neurons and glia also produced atleast one other cell type that did not express any of thedifferentiation markers tested; the identity of these cells is unknown.Taken together, these data establish the multipotency of the rat neuralcrest cell identified and isolated by virtue of co-expression of LNGFRand nestin.

EXAMPLE 4 Self-renewal of Multipotent Neural Crest Cells in vitro

After 10 days in culture in medium supplemented with 10% CEE and on aFN/PDL substrate, all of the neural crest cell clones that containedneurons also contained non-neuronal cells expressing LNGFR and nestin(as described above). In order to determine whether these cells wereimmature glia, or multipotent neural crest cells that had undergoneself-renewal, serial subcloning experiments were performed. FIG. 7provides a flow chart summarizing these serial subcloning experiments.In FIG. 7, "plating medium" refers to complete medium containing CEE andlacking FCS and "differentiation medium" refers to SCD medium containingFCS and forskolin.

For serial sub-cloning experiments, clones were harvested and replatedas follows. The primary clones were examined microscopically to ensurethat there were no impinging colonies and that the whole clone fitswithin the inscribed circle. Using sterile technique throughout theprocedure, glass cloning cylinders (3 mm id.) were coated on one endwith silicone grease (Dow Corning) and placed about the primary clone sothat the grease formed a seal through which medium could not pass. Thecells were removed from the cylinder by first treating them with 100 mlof 0.05% Trypsin solution (Gibco) for 3 minutes at 37° C. in ahumidified 5% CO₂ incubator. At room temperature 70 μl of the trypsinsolution was removed and replaced with 70 μl of complete medium. Thecells were resuspended into the 100 μl volume by vigorous triturationthrough a pipet tip and the whole volume was diluted into 5 ml ofcomplete medium. The 5 ml was then plated onto 1 or 2 60 mm dishes whichwere placed in a humidified 5% CO₂ incubator for 2 hours at which timethe medium was exchanged for fresh complete medium. Single founderscells were then identified and allowed to grow into secondary clones asdescribed below.

Primary clones founded by LNGFR-positive progenitor cells were allowedto row for 6 days (FIG. 8, Panel A) on a PDL/FN substrate. At this time,clones containing LNGFR-positive cells were identified by live cellsurface labeling, and these clones were then removed from their originalplates by trypsinization, as described above. The dissociated cells werethen replated at clonal density under the same culture conditions astheir founder cells. Individual secondary founder cells were identifiedby labeling live cells with 192-Ig and their positions marked (FIG. 8,Panels B and B' show two individual secondary founder cells; Panels Cand C' show the clonal progeny of these individual cells at day 17).Both non-neuronal, neurite bearing cells are visible in the clones (FIG.8, panels C and C').

A clone derived from secondary founder cells, such as that shown in FIG.8, was transferred into SCD medium to allow the expression of Schwanncell markers. After approximately 10 days, the subdlone was fixed, anddoublelabeled for NF160 and GFAP (FIG. 9, Panel A shows the clone inphase contrast; Panel B shows labeling with anti-NF160; Panel C showslabeling with anti-GFAP). The apparent labeling of neurons in panel C isan artifact due to bleed-through into the fluorescein channel of theTexas Red fluorochrome used on the goat anti-rabbit secondary antibodyin panel B.

Additionally, following 10 days of secondary culture, living subcloneswere scored visually for the presence of neurons and glia by doublelabeling with 192-Ig (anti-LNGFR) and 5A5, a monoclonal antibody to highPSANCAM.

Single neural crest cells isolated from primary clones were able toproliferate and generate clones containing both neurons and non-neuronalcells, probably glia. Quantitative analysis of clones derived from 16different primary and 151 secondary founders after ten days in platingmedium indicated that over 30% of the total secondary founder cells gaverise to clones containing neurons (N), glia (G) and other (0) cells(Table I, N+G+O). Of the remaining 70% of the founder cells, however,almost 50% failed to form clones and died; thus of the clonogenic (i.e.,surviving) founders, 54% were of the N+G+O type (Table I). To confirmthat these mixed clones indeed contained glia or glial progenitors, theywere transferred to SCD medium and allowed to develop for an additional7 days, then fixed and double-stained for neurofilament and GFAPexpression. As was the case for the primary clones, this treatmentcaused expression of GFAP in a high proportion of non-neuronal cells inthe clones (FIG. 9), confirming the presence of glia. These dataindicate that primary neural crest cells are able to give rise at highfrequency to progeny cells retaining the multipotency of theirprogenitors, indicative of self renewal. However, in several casessecondary clones containing only neurons were found (Table I, N only),and many of the secondary clones contained glia and other cells but notneurons (Table 1, G+O). This observation suggests that in addition toself-renewal, proliferating neural crest cells may undergo lineagerestriction in vitro as well to give rise to glial or neuronalprogenitor cells which are characterized by the capacity to divide andself-renew but are restricted to either the neuronal or glial lineage.

                  TABLE I                                                         ______________________________________                                        Sub-Clone Phenotype total # (%)                                                                                            No                                                                             Primary # of 2°  N +                                                    clone                            Clone ID Founders N only G + O G + O O found                                ______________________________________                                        1.1    21       0       15 (71)                                                                               0     0     6 (29)                              1.18 6 0  1 (17)  1 (17)  2 (33)  2 (33)                                      1.24 5 1 (20)  0  1 (20)  2 (40)  1 (20)                                      2.6 7 0  0  1 (14)  1 (14)  5 (72)                                            2.18 7 0  0  1 (14)  0  6 (86)                                                3.14 20  0  2 (10)  4 (20)  0 14 (70)                                         3.18 4 0  1 (25)  0  0  3 (75)                                                4.5 1 0  1 (100)  0  0  0                                                     4.8 9 0  0  1 (11)  2 (22)  6 (67)                                            4.14 10  0  2 (20)  3 (30)  1 (10)  4 (40)                                    5.2 15  1 (7)  8 (53)  0  0  6 (40)                                           6.1 13  0  2 (15)  2 (15)  0  9 (70)                                          6.2 17  1 (6)  2 (12)  4 (24)  0 10 (58)                                      6.17 2 0  1 (50)  0  0  1 (50)                                                8.2 5 0  4 (80)  0  0  1 (20)                                                 8.5 9 0  4 (44)  0  0  5 (56)                                                 Mean ±                                                                     s.e.m.                                                                        % total  2.1 ± 31 ± 10 ± 7.4 ± 49 ±                            founders  1.3 7.9 2.6 3.3 6                                                   % clono-  3.1 ± 54 ± 29 ± 15 ±                                    genic  1.8 11 8 6                                                             founders                                                                    ______________________________________                                    

EXAMPLE 5 Substrate Composition Influences the Developmental Fate ofMultipotent Neural Crest Cells

The foregoing experiments indicate that neural crest cells grown on aPDL/FN substrate generate clones containing both peripheral neurons andglia. When the same cell population is grown at clonal density on asubstrate containing FN only, the resulting clones contain glia and"other" cells but never neurons (FIGS. 10 and 11, Panels D,E,F). FIG. 10provides a flow chart summarizing the following experiments whichdemonstrate the substrate effect on the fate of mammalian neural crestcells. FIG. 11 shows the immunoreactivity of cells stained for variousmarkers.

On FN alone, G+O clones are obtained containing non-neuronal cellsexpressing high levels of LNGFR immunoreactivity, but neither NCAM⁺ norneurite-bearing cells (FIG. 11, panels E,F). By contrast on PDL/FN, theclones contain both LNGFR⁺, NCAM⁻ non-neuronal crest cells and LNGFR⁻,NCAM⁺ neurons (FIG. 11, panels B,C). Quantification indicated that on FNalone, 70-80% of the clones are of the G+O phenotype and none of theN+G+O phenotype (FIG. 12, panel A), whereas on PDL/FN 60% of the clonesare of the N+C +O and only 20% are of the GAO phenotype (FIG. 12, panelB). These data indicate that the composition of the substrate affectsthe phenotype of neural crest cells that develop in culture.

To rule out the possibility that the foregoing results could beexplained simply by the failure of neurogenic crest cells to adhere andsurvive on a FN substrate, a different experiment was performed in whichall the crest cells were initially cloned on a FN substrate. FIG. 13provides a flow chart summarizing these experiments. These experimentswere performed to demonstrate that differences in attachment and/orsurvival do not account for differences in eventual clone composition.Subsequently, one group of cells was exposed to PDL as an overlay inliquid media (0.05 mg/ml) after 48 hrs, while a sister culture wasretained on FN alone as a control (FIG. 13). Clones expressing LNGFRwere identified by live cell surface labeling at the time of the PDLoverlay and the development of only LNGFR⁺ clones was further monitored.After two weeks, the cultures were transferred to SCD medium for anadditional 10 days of culture, and their phenotypes then scored aspreviously described.

By contrast to clones maintained on FN, where no neurons developed, manyof the clones exposed to a PDL overlay contained neurons at the end ofthe culture period (FIG. 14, panel A). Moreover, virtually none of theclones were of the G+O phenotype after the PDL overlay. These dataindicate that an overlay of PDL is able to alter the differentiation ofneural crest cells even if they are initially plated on an FN substrate.Moreover, they suggest that at least some of the N+G+O clones derived byconversion of founder cells that would have produced G+O clones on FN.However, because of the increased cytotoxicity obtained from the PDLoverlay, it was not possible to rule out the possibility that many ofthe cells that would have produced G+O clones simply died. To addressthis issue, the PDL overlay was performed on a parallel set of culturesat day 5 rather than at 48 hrs. Under these conditions, virtually all ofthe LNGFR⁺ clones survived and differentiated. 60% of these clonescontained neurons, whereas 35% contained GAO (FIG. 14, panel B). Bycontrast, greater than 90% of the clones maintained on FN developed to aG+O phenotype. Since little or no clone death was obtained under theseconditions, and since a majority of the clones contained neuronsfollowing the PDL overlay at day 5, these data suggest that PDL convertspresumptive G+O clones into N+G+O clones. However the fact that 35% ofthe clones became G+O following PDL overlay at days, whereas virtuallynone did so when the overlay was performed at 48 hrs (FIG. 14, compareG+O, hatched bars, in panels A and B), suggests that some clones mightbecome resistant to the effect of PDL between 48 hrs and days.

EXAMPLE 6 Substrate Influences Latent Developmental Potential of NeuralCrest Cells

To demonstrate more directly that the substrate can alter thedevelopmental fate of neural crest cells, a serial subcloning experimentwas performed. Clones were established on FN, and after 5 days theprogeny of each clone were subdivided and cloned onto both FN and PDL/FNsubstrates. Following 10 days of culture in standard medium, the cloneswere shifted to SCD medium for an additional week to ten days and thenfixed, stained and scored for the presence of neurons and Schwann cells.Five of seven primary clones founded on FN gave rise to secondary clonescontaining neurons when replated onto a PDL/FN substrate at days (TableII). On average, 57±17% of the secondary clones contained neurons. Bycontrast, none of the sister secondary clones replated onto FN containedneurons (Table II). These data confirm that the PDL/FN substrate is ableto alter the fate of neural crest cell clones initially grown on FN.They also reveal that the "neurogenic potential" of neural crest cellsis retained, at least for a period of time, on FN even though overtneuronal differentiation is not observed. This suggested that FN isnon-permissive for overt neuronal differentiation under these cultureconditions. In support of this idea, when primary clones established onPDL/FN were replated onto FN, none of the secondary clones containedneurons, whereas 100% (5/5) of the primary clones gave rise toneuroncontaining secondary clones when replated onto PDL/FN (Table II).Moreover, on average 93±7% of the secondary clones derived from eachprimary clone contained neurons on PDL/FN, indicating that most or allof the clonogenic secondary crest cells retained neurogenic potentialunder these conditions.

While this experiment indicated that at least some neural crest clonesretain neurogenic potential on FM, not all clones exhibited thiscapacity. This could indicate a heterogeneity in the clonogenic foundercells that grow on FN, or it could indicate a progressive loss ofneurogenic potential with time in culture on FM. To address this issue,a second experiment was performed in which primary clones were replatedat day 8 rather than at day 5. In this case, a more dramatic differencewas observed between primary clones established on FM versus on PDL/FN.Only 1/6 primary FM clones replated at day 8 gave rise to any secondaryclones containing neurons on PDL/FN, and in this one case only 17% ofthe secondary clones contained neurons (Table II). By contrast, 6/6primary PDL/FN clones gave rise to neuron-containing secondary cloneswhen replated on PDL/FN at day 8, and 52+7% of these secondary clonescontained neurons (Table II). These data suggest that neurogenicpotential is gradually lost by neural crest cells cultured on FM, butretained to a much greater extent by the same cells grown on PDL/FN.Thus the composition of the substrate influences not only the overtdifferentiation of the neural crest cells, but also their ability tomaintain a latent developmental potential over multiple cellgenerations.

                  TABLE II                                                        ______________________________________                                        1° Substrate                                                                    FN            %       pDL/FN    %                                    2° Substrate                                                                    FN    pDL/FN  Neuronal                                                                              FN  pDL/FN                                                                              Neuronal                             ______________________________________                                        Day 5    0/7   5/7     57 ± 17                                                                            0/5 5/5   93 ± 7                              Replating                                                                     Day 8 0/6 1/6 17 0/6 6/6 52 ± 7                                            Replating                                                                   ______________________________________                                    

EXAMPLE 7 Identification of Neural Crest Stem Cells by Transplantation

Neural crest stem cells are identified by two general criteria: by theirantigenic phenotype, and by their functional properties. Thesefunctional properties may be assessed in culture (in vitro), asdescribed above, or they may be assessed in an animal (in vivo). Theabove examples described how the self-renewal and differentiation ofneural crest stem cells can be assayed in vitro, using clonal cellcultures. However, these properties may also be determined bytransplanting neural crest cells into a suitable animal host. Such anassay requires a means of delivering the cells and of identifying thetransplanted cells and their progeny so as to distinguish them fromcells of the host animal. Using standard techniques, it is possible todeliver neural crest cells to a developing mammalian or avian embryo orto any tissue or compartment of the adult animal (e.g., brain,peritoneal cavity, etc.).

For example, neural crest cell cultures are prepared as describedearlier. After a suitable period in primary or secondary culture, neuralcrest cells are identified by live cell-labeling with antibodies toLNGFR, and removed from the plate using trypsin and a cloning cylinder,as described in previous examples. The cells are diluted intoserum-containing medium to inhibit the trypsin, centrifuged andresuspended to a concentration of 10⁶ -10⁷ cells per milliliter. Thecells are maintained in a viable state prior to injection by applyingthem in small drops (ca. 10 μl each) to a 35 mm petri dish, andevaporation is prevented by overlaying the droplets with light mineraloil. The cells are kept cold by keeping the petri dishes on ice. Forinjections into mouse embryos, pregnant mothers at embryonic day 8.5-9.0are anaesthetized and their uterus exposed by an incision into theabdomen. Neural crest cells are drawn into a sharpened glassmicropipette (with a sealed tip and hole in the side to prevent cloggingduring penetration of tissues) by gentle suction. The pipette isinserted into the lower third of the deciduum and a volume ofapproximately 0.5 μl is expelled containing approximately 1000 cells.The micropipette is withdrawn and the incision is sutured shut. After anadditional 3-4 days, the mother is sacrificed, and individual embryosare removed, fixed and analyzed for the presence and phenotype of cellsderived from the injected neural crest cells.

To identify the progeny of the injected cells, it is necessary to have ameans of distinguishing them from surrounding cells of the host embryo.This may be done as follows: rat neural crest cells are injected into amouse embryo (following suitable immunosuppression of the mother orusing a genetically immunodeficient strain such as the SCID strain ofmice), the injected cells are identified by endogenous markers such asThyl or major histocompatibility complex (MHC) antigens using monoclonalantibodies specific for the rat Thyl or MHC antigens. Alternatively, anexogenous genetic marker is introduced into the cells prior to theirtransplantation as a means of providing a marker on or in the injectedcells. This is as follows: neural crest cells in culture are incubatedwith a suspension of replication-defective, helper-free retrovirusparticles harboring the lacZ gene, at a titer of 10⁵ -10⁶ pfu/ml in thepresence of 8 μl/ml polybrene for four hours. The cells are then washedseveral times with fresh medium and prepared for injection as describedabove. The harvested embryos are then assayed for expression ofP-galactosidase by whole mount staining according to standardprocedures. The blue cells (indicating expression of the lacZ gene) willcorrespond to the progeny of the injected neural crest cells. Thisprocedure can be applied to any tissue or any stage of development inany animal suitable for transplantation studies. Following whole-mountstaining, embryos bearing positive cells are embedded in freezing mediumand sectioned at 10-20 μm on a cryostat. Sections containing blue cellsare selected, and then counterstained for markers of mature neurons andglia using specific antibodies, according to standard techniques, andimmunoperoxidase or alkalinephosphatase histochemistry. Theidentification of lacZ+ (blue) cells expressing neuronal or glialmarkers indicates that the progeny of the injected neural crest cellshave differentiated appropriately. Thus, this technique provides a meansof identifying mammalian neural crest stem cells through transplantationstudies to reveal the function of said stem cells.

EXAMPLE 8 Genetic-Engineering of Neural Crest Stem Cells (NCSCs)

A. Retroviral infection of NCSCs

In this method, NCSCs are infected with a replication-incompetent,recombinant retrovirus harboring the foreign gene of interest. Thisforeign gene is under the control of the long terminal repeats (LTRs) ofthe retrovirus, in this case a Moloney Murine Leukemia Virus (MoMuLv)(Cepko et al. (1984) Cell 37:1053-1062). Alternatively, the foreign geneis under the control of a distinct promoter-enhancer contained withinthe recombinant portion of the virus (i.e., CMV or RSV LTR). In thisparticular example, the E. coli β-galactosidase gene was used, becauseit provides a blue histochemical reaction product that can easily beused to identify the genetically-engineered cells, and thereby determinethe transformation efficiency.

Rat NCSC cultures were established as described above. Twenty-four hoursafter replating, the cells were exposed to a suspension ofβ-galactosidase-containing retrovirus (Turner et al. (1987) Nature328:131-136) with a titer of approximately 10⁵ -10⁶ pfu/ml in thepresence of 8 μg/ml polybrene. Following a 3 hr exposure to the viralsuspension, the cultures were rinsed and transferred into standardmedium. After three days of growth in this medium, the transformed cellswere visualized using the X-gal histochemical reaction (Sanes et al.(1986) EMBO J. 5:3133-3142) FIG. 15, Panel A shows the NCSC culturethree days after infection with the lacZ containing retrovirus, afterfixation and staining using the X-gal reaction.β-galactosidase-expressing cells are indicated by the solid arrows.Non-expressing cells in the same microscopic field are visualized byphase contrast microscopy (B), and are indicated by open arrows. Theblue, β-galactosidase⁺ cells represented approximately 5-10% of thetotal cells in the culture as visualized by phase-contrast microscopy(FIG. 15, Panel B).

B. Calcium-Phosphate-Mediated Transfection of NCSCs

In this method, NCSCs are transfected with an expression plasmid usingthe calcium phosphate method (Wigler et al. (1979) Proc. Natl. Acad.Sci. USA 76:1373-1376). As in the previous example, the β-galactosidasegene was used to facilitate visualization of the transfected cells.

In this case, the vector pRSVlacZ was used, in which the β-galactosidasegene (lacZ) is under the control of the Rous Sarcoma Virus (RSV) LTR,and the SV40 intron and poly A-addition site are provided at the 3' endof the gene (Johnson et al. (1992) Proc. Natl. Acad. Sci. USA89:3596-3600).

NCSCs were established in 35 mm tissue culture dishes. 24 hr afterplating, a calcium phosphate precipitate containing approximately 20μg/ml of pRSVlacZ was prepared. 123 μl of this precipitate was added toeach dish, and incubated at room temperature for 20 minutes. Two ml ofstandard medium containing 30 μM chloroquine was then added to each dishand incubation was continued overnight at 37° C. The next day, themedium was replaced and incubation continued for a further two days. Thecultures were then fixed and assayed for β-galactosidase expression bythe standard X-gal reaction. Approximately 10% of the NCSCs expressedthe lacZ reaction product.

C. Immortalization of NCSCs

NCSC cultures are established as described above. The cultures areexposed, in the presence of 8 μg/ml polybrene, to a suspension ofretrovirus harboring an oncogene preferably selected from theimmortalizing oncogenes identified herein. These retroviruses contain,in addition to the oncogene sequences, a gene encoding a selectablemarker, such as hisD, driven by the SV40 early promoter-enhancer(Stockschlaeder, M. A. R. et al. (1991) Human Gene Therapy 2:33). Cellswhich have taken up the hisD gene are selected for by growth in thepresence of L-histidinol at a concentration of 4 mM. Alternatively,selection can be based upon growth in the presence of neomycin (500μg/ml). NCSCs are infected with the above retroviruses which areconcentrated to a titer of greater than 10⁶ pfu/ml by centrifugation.The virus is applied to the cells in two sequential incubations of 4-8hours each in the presence of 8 μglml polybrene.

Following infection, the cells are grown in the presence of 4 mML-histinol or 500 μg/ml neomycin (G418) for 5-10 days. Cells whichsurvive the selection process are screened for expression of LNGFR bylive-cell labeling using the monoclonal antibody 192 Ig as describedabove. Colonies containing a homogeneous population of LNGFR+ cells arecloned using a cloning cylinder and mild trypsinization, and transferredinto duplicate FN/pDL-coated 96-well plates. After a short period ofgrowth, one of the plates is directly frozen (Ramirez-Solis, R. et al.(1992) Meth. Enzymol., in press). The cells in the other plate arereplated onto several replicate 96-well plates, one of which ismaintained for carrying the lines. The cells on the other plates arefixed and analyzed for the expression of antigenic markers. Successfulimmortalization is indicated by (1) the cells homogeneously maintain anantigenic phenotype characterized by LNGFR+, nestin+, lin-(where "lin"refers to lineage markers characteristic of differentiated neuronal orglial crest derivatives, including neurofilament, peripherin, hiPSA-NCAM, GFAP, O₄ and P_(o)); and (2) the cell population isphenotypically stable over several weeks of passage (as defined by lackof differentiation to morphologically- and antigenically-recognizableneurons and/or glia). The ability of the lines to differentiate istested by transferring them to conditions that promote differentiation(omission of CEE in the case of neurons and addition of serum and 5 μMforskolin for Schwann cells). Maintenance of the ability todifferentiate is a desirable, although not necessary, property of theconstitutively-immortalized cells.

EXAMPLE 9 Generation of Monoclonal Antibody to Mouse LNGFR

Mouse monoclonal antibodies specific to LNGFR from primates (Loy et al.(1990), J. Neruosci. Res. 27:657-664) and rat (Chandler et al. (1984) J.Biol. Chem. 259:6882-6889) have been produced. No monoclonal antibodiesto mouse LNGFR have been described. We have produced rat monoclonalantibodies to mouse LNGFR. These antibodies recognize epitopes presenton the surface of living cells such as Schwann cells, making themsuitable for use in immunologic isolation of multipotent neural stemcells (such as neural crest stem cells) and their differentiatedderivatives (as well as neural progenitor cells from the CNS) frommurine species. The isolation of such cells from mice is particularlydesirable, as that species is the experimental organism of choice forgenetic and immunological studies or human disease.

To generate monoclonal antibodies to mouse LNGFR, a genomic DNA fragmentencoding the extracellular domain (ligand binding domain) of thatprotein was expressed in E. coil, as a fusion protein withglutathione-Stransferase (Lassar et al. (1989) Cell 58:823-831).Briefly, a probe for the extracellular domain based on either of theknown DNA sequences for rat and human LNGFR is used to screen a mousegenomic library. A cloned insert from a positively hybridizing clone isexcised and recombined with DNA encoding glutathione with appropriateexpression regulation sequences and transfected into E. coli. The fusionprotein was affinity-purified on a glutathione-Sepharose column, andinjected into rats. Sera obtained from tail bleeds of the rats werescreened by surface-labeling of live Schwann cells isolated from mousesciatic nerve by standard procedures (Brockea et al. (1979) In Vitro15:773-778. Surface labeling was with labelled goat anti-rat antibodyFollowing a boost, fusions were carried out between the rat spleen cellsand mouse myeloma cells. Supernatants from the resulting hybridomacultures were screened using the live Schwann cell assay. Positiveclones were re-tested on NIH 3T3 fibroblasts, a mouse cell line thatdoes not express LNGFR, and were found to be negative. The use of thislive cell assay ensures that all antibodies selected are able torecognize LNGFR on the surface of living cells. Moreover the assay israpid, simple and more efficient than other assays such as ELISA, whichrequire large quantities of purified antigen.

Approximately 17 independent positive hybridoma lines were identifiedand subcloned. An example of the results obtained with the supernatantfrom one such line 19 shown in FIG. 16. A culture of mouse sciatic nerveSchwann cells was labeled with one of the rat anti-mouse LNGFRmonoclonal antibodies and counterstained with DAPI to reveal the nucleiof 611 cells. The left panel (A) shows that most of the cells arelabeled on their surface with the anti-LNGFR antibody (red staining;solid arrows), the right panel (B) reveals all the cell nuclei on theplate, and shows a few cells not labeled by the anti-LNGFR antibody(blue staining; open arrows; compare to left panel). These unlabeledcells most likely represent contaminating fibroblasts which are knownnot to express LNGFR. These cells provide an internal control whichdemonstrates the specificity of the labeling obtained with theanti-LNGFR antibody.

EXAMPLE 10 O Cells are Smooth Muscle Cells

To determine whether O cells could be smooth muscle ("SM") cells,cultures of neural crest cells containing these cells were stained witha monoclonal antibody to smooth muscle actin ("SMA"), a marker of smoothmuscle cells (Skalli et al (1966) J. Cell Biol. 103:2787-2796). Thecultures were counter-stained with anti-p75 to identify the neural creststem cells. The anti-SMA antibody labeled a significant number of cells(FIG. 17B, open arrows), and these cells did not express p75 on theirsurface and were clearly distinct from the p75-expressing neural creststem cells (FIG. 17B, closed arrow). However, clonal analysis indicatedthat both p75⁺, SMA⁻ cells and p75⁻, SMA⁺ cells derived from a p75⁺neural crest stem cell progenitors (see below).

To establish that individual neural crest stem cells could generateneurons, glia and smooth muscle cells, a clonal analysis was performed.Individual p75+ neural crest stem cells were identified and allowed todevelop for two weeks in culture. The resultant clones were then fixedand triply-labeled with antibody to peripherin (to detect neurons), GFAP(to detect glia) and SMA (to detect smooth muscle cells). As shown inFIG. 18, within the same clone it was possible to identify neurons(FIGS. 18A, 18B, arrowhead), glia (FIGS. 18C, open arrows) and smoothmuscle cells (FIG. 18C, closed arrow), confirming that the neural creststem cell is able to generate all three lineages in our culture system.

The foregoing experiments were carried out in standard medium lackingfetal bovine serum. Previously, we observed that the addition of fetalbovine serum to this medium at early times of culture resulted in theextinction of LNGFR expression. Taken together with the foregoingobservation that SMA⁺ cells are LNGFR⁻, we asked whether cells grown instandard medium+fetal bovine serum expressed smooth muscle markers. Theresults indicate that virtually all cells obtained in SM+fetal bovineserum express high levels of SMA (FIGS. 20A, 20B). To further establishtheir identity as smooth muscle cells, these cells were also stainedwith two other markers of smooth muscle: desmin (Lazarides, et al (1978)Cell 14:429-438) and calponin (Gimona et al (1990) FEBS Lett.274:159-162). The SMA+cells were also labeled by anti-desmin antibody(FIG. 3C) and by anti-calponin (FIGS. 3A, B). These data confirm thatthe O cells are indeed smooth muscle cells, and also show that fetalbovine serum contains one or more substances able to drive virtually allneural crest stem cells into the smooth muscle lineage.

Differentiated smooth muscle cells have been isolated and cultured fromthe vasculature, for example, Chamley-Campbell et al (1990) Phys. Rev.59:161, but previously it has not been possible to obtain the de novodifferentiation of such cells from an undifferentiated progenitor. Thedata presented above identify neural crest stem cells as progenitors ofsmooth muscle, as well as of neurons and glia, and indicate that theycan be induced to differentiate to smooth muscle in culture using fetalbovine serum. Such differentiation occurs at the expense of neuronal andglial differentiation, which does not occur in the present of fetalbovine serum (Stemple et al. (1992), Cell 71:973-985. Thus, neural creststem cells should be useful for identifying smooth muscledifferentiation factors present in fetal bovine serum, as well as foridentifying other growth, survival or differentiation factors for smoothmuscle present in other sources.

EXAMPLE 11 Neural Crest Stem Cells Preferentially Differentiate toNeurons or Smooth Muscle Cells

NCSCs grown at clonal density in standard culture medium undergosymmetrical, self-renewing divisions for at least 5-6 days in vitro.Stemple and Anderson (1992) Cell 71:973-985. Neurons do not begin todifferentiate in such cultures until 10-15 days of incubation. Moreover,clones containing only neurons are never observed; rather the neuronsdifferentiate together with nonneuronal cells such as glia. Stemple andAnderson (1992) Cell 71:973-985.

To determine if and how neural crest stem cells respond to growthfactors from the TGF-β superfamily neural crest stem cells were grown in1.6 nM recombinant bone morphogenic protein 2 ("rBMP2"). Manyneuron-only colonies (identified by their neurite-bearing morphology andexpression of peripherin) developed within 3-4 days (FIGS. 21A and 21B).At this dose, ˜50% of the colonies contained only neurons; 20%-25%contained neurons (about as many per colony as in the neuron-onlycolonies) as well as large flat cells; the remainder consisted only ofsuch flat cells. Thus, 75% of colonies grown in rBMP2 contained neuronsafter 4 days. By contrast, none of the colonies grown in the absence ofrBMP2 contained any neurons at this time point (FIGS. 21E and 21F).Glial cells were not detected at any time in BMP2-containing cultures.The phenotype of the large, flat cells is described below. Comparableresults were obtained using rBMP4 (data not shown), which is known tohave virtually indistinguishable biological activities from BMP2 in mostassays examined. Kingsley (1994) Genes Dev. 8:133-146.

Most of the neurons that developed in rBMP2 stained positively withmonoclonal antibody B2 (FIG. 21D), which is only expressed by autonomicneurons. Anderson et al. (1991) N. Neurosci. 11:3507-3519. However,these neurons did not express catecholamine biosynthetic enzymes such asdopamine β-hydroxylase or tyrosine hydroxylase, at any concentration ofthe factor tested. These data suggest that rBMP2 promotes thedifferentiation of autonomic neurons, which are either nonsympathetic orwhich require additional signals (Groves et al.(1995) Development121:887-901) to express markers characteristic of the sympatheticsublineage (for review, see Patterson and Nawa (1993) Cell 72/Neuron10(Suppl.):123-137).

As overall neuronal differentiation was not apparent until 3-4 daysafter addition of rBMP2, we sought evidence for an earlier influence ofthis factor on neurogenesis. To do this, we examined expression ofMASH1, whose expression precedes that of neuronal markers by severaldays both in vivo (Lo et al.(1991) Genes Dev. 5:1524-1537) and in vitro(Shah et al. (1994) Cell 77:349-360). At 12 hr after addition of rBMP2to NCSCs, over 70% of the colonies (many of which were still singlecells) expressed MASH1 (FIGS. 25A and 25B); by 24 hr, ˜85% of thecolonies were MASH1. The effects of rBMP2 were apparent even by 6 hr,the earliest time tested, when ˜30% of the colonies expressed MASH1. Bycontrast, at these time points very few of the NCSCs in control mediumwere MASH1 (FIGS. 25C and 25D). Rather, MASH1 is expressed by NCSCs incontrol cultures only after 7-8 days (Shah et al. (1994) Cell77:349-360). Moreover, within such control colonies, MASHI is expressedby subsets of cells; by contrast, within rBMP2-treated colonies most orall cells expressed MASH1. These data indicate that in the presence ofrBMP2 the majority of NCSCs express MASH1, and do so on a far earlierschedule than under controlled conditions. Shah et al.(1996) Cell85:331-343. Moreover, they support the idea that the expression of MASH1in autonomic neuronal precursors in vivo may reflect its induction byBMP2 derived from neighboring tissues. Shah et al.(1996) Cell85:331-343.

A subset of the colonies in rBMP2 also contained large, flat cells thatsuggested they could be a mesectodermal derivative of the neural crest,such as smooth muscle (Chamley-Campbell et al.(1979) Physiol. Rev.59:1-61; Ito and Shah et al.(1996) Cell 85:331-343. Many of the flatcells expressed αSMA, a well-characterized SM marker (Owens (1995)Physiol. Rev. 75:487-517) (FIG. 22C, green fluorescence, and data notshown). Further, most of these flat cells expressed calponin, anotherSM-specific protein that may regulate contractility (Owens, 1995) (FIG.22C, red fluorescence). Of all nonneuronal cells observed in rBMP2, 93%expressed αSMA, calponin, or both. The remaining cells displayed asimilar SM-like morphology (FIG. 22A) despite their lack of expressionof these two SM-specific markers. These data therefore suggest that mostor all of the flat cells observed in rBMP2 are SM cells are variousstages of differentiation.

Other growth factors from the TGF-β superfamily were screened for theireffects on neural crest stem cells. In recombinant TGFβ1 ("rTGFβ1")virtually all neural crest stem cell colonies differentiated to SM cells(FIG. 22D). Of the colonies, 82.4%±0.6% (mean±SEM, n=2) consistedexclusively of cells that were αSMA⁺, calponin⁻, or both (FIG. 22F; 12%has at least one αSMA⁺ or calponin⁺ cell together with SM-like,markernegative cells, while 5.6%±1.8% of the colonies contained onlymarkernegative but SM-like cells. Less than 1% of the colonies containedany low affinity nerve growth factor receptor-positive (LNGFR⁺) NCSCs.No neurons or glial cells were observed to develop under theseconditions. In cultures grown in the absence of TGFβ1 for a similarperiod, 95% of the colonies consisted primarily of undifferentiatedNCSCs, although some SM cells were present. TGFβ2 and TGFβ3 yieldedsimilar results as TGFβ1 (data not shown).

The fact that BMP2 produced neurons and some SM cells, while TGFβ1produced only SM cells, could simply reflect the differentconcentrations at which these related factors were initially used.However, dose-response experiments (FIG. 23) did not support this idea;there were no doses at which the factors elicited identical responses,or at which BMP2 elicited a homogeneous response. Thus, exclusively SMor neuronal differentiation was not observed at any concentration BMP2;rather, the proportion of both neuronal and SM colonies increased as afunction of BMP2 dose (FIGS. 23A and 23B). Similarly, varying theconcentration of TGFβ1 over three orders of magnitude did not cause theappearance of mixed (i.e., neuronal+SM) colonies at any dose (FIG. 23Cand data not shown). These data suggest that the mixed response observedwith BMP2 cannot be explained by a suboptimal or excess concentration ofthe factor used.

EXAMPLE 12 Instructive (Not Selective)effect of BMP2 and TGF-β on NeuralCrest Stem Cells

A clonal analysis was performed in order to distinguish whether BMP2 andTGFβ1 act to influence differentiation by multipotent neural crest stemcells, or rather to support survival of subpopulations of pre-committedneuronal or SM precursors, respectively. Individual NCSCs wereidentified shortly after plating, growth factors were added to some, andtheir subsequent survival and differentiation assessed after 4 days.Selective survival of subsets of clones was not observed. Dailyobservation of the cultures indicated that none of them containedneurons prior to death; in fact, many contained cells with a SM-likemorphology. These observations indicated that in the presence of rBMP2or TGFβ1, multipotent neural crest cells that would eventually havegenerated neurons, glia, and SM cells (in control medium) insteadgenerated only neurons and SM cells, or SM cells alone, such that BMP2and TGFβ1 acted instructively on the founder cell population. Shah etal.(1996) Cell 85:331-343. In order to exclude the possibility thatthese factors were acting selectively on the clonal progeny of thefounder cells, sequential observations of live, identified clones, weremade every 24 hr for the 4-day incubation period (FIG. 24). Significantcell death did not within clones in either the presence or absence ofthe growth factors. To the contrary, in rBMP2, many instances wereobserved in which a founder cell divided several times and all of itsprogeny then differentiated into neurons (FIG. 24B). Similarly, inrTGFβ1 many cases were documented in which a founder cell divided toproduce a clone of SM-like cells without any noticeable cell death (FIG.24C). The behavior of the clones in rBMP2 and rTGFβ1 was in clearcontrast to that observed in control medium over the same cultureperiod, in which neural crest stem cells divided to produce clonescontaining neural crest stem cell-like cells (FIG. 24A). Stemple andAnderson, (1992) Cell 71:973-985. No death of SM-like or othernonneuronal cells within neuron-only clones that developed in rBMP2 wasdetected, by criteria of either pycnotic nuclei or cell carcasses(usually visible on the substrate following death). Moreover, neuronswere never observed to differentiate and then die, in either controlmedium or in TGFβ1.

The results from serial observation indicate reduced clone size in TGFp1and rBMP2 which is most likely due to inhibited or slowed proliferation.Whether such effects on proliferation are a cause or a consequence ofdifferentiation remains to be determined; however, TGFβ1 is known toinhibit proliferation of SM cells in low density cultures. (Majack(1987) J. Cell Biol. 105:465-471.

Although neurons eventually differentiate in the absence of rBMP2, thisfactor not only accelerates neurogenesis: half of the clones grown inrBMP2 contain only neurons; by contrast no such clones are ever observedin control conditions. Sequential observation of individual clones (FIG.24) makes it unlikely that this is due to the interclonal death ofnonneuronal cells that are initially generated despite the presence ofrBMP2.

What is claimed is:
 1. A method for producing a population of mammalianneurons or smooth muscle cells comprising contacting in vitro at leastone mammalian neural stem cell with a growth factor wherein said growthfactor is a member of the transforming growth factor-β ("TGF-β")superfamily of growth factors.
 2. The method of claim 1, wherein saidpopulation of cells produced comprises mammalian neurons.
 3. The methodof claim 2, wherein said growth factor is bone morphogenic protein 2("BMP-2").
 4. The method of claim 2, wherein said growth factor is bonemorphogenic protein 4 ("BMP-4").
 5. The method of claim 2, wherein saidstem cell comprises a neural crest stem cell.
 6. The method of claim 1,wherein said population of cells produced comprises mammalian smoothmuscle cells.
 7. The method of claim 6, wherein said growth factor isTGF-β-1.
 8. The method of claim 6, wherein said growth factor isTGF-β-2.
 9. The method of claim 6, wherein said growth factor isTGF-β-3.
 10. The method of claim 6, wherein said growth factor is bonemorphogenic protein 2 ("BMP-2").
 11. The method of claim 6, wherein saidgrowth factor is bone morphogenic protein 4 ("BMP-4").
 12. The method ofclaim 6, wherein said stem cell comprises a neural crest stem cell. 13.The method of claim 2, further comprising detecting the differentiationof said stem cell to said population of mammalian neurons.
 14. Themethod of claim 13, further comprising detecting the differentiation ofsaid stem cell, wherein said population of neurons are autonomicneurons.
 15. The method of claim 14, wherein said detecting is with anantibody specific for a marker for autonomic neurons.
 16. The method ofclaim 14, wherein said detecting is with antibody B2, specific for amarker for autonomic neurons.
 17. The method of claim 15, wherein saiddetecting is with the antibody specific for neurofilament.
 18. Themethod of claim 15, wherein said detecting is with the antibody specificfor peripherin.
 19. The method of claim 6, further comprising detectingthe differentiation of said stem cell to said population of smoothmuscle cells.
 20. The method of claim 19, wherein said detecting is withan antibody specific for a marker for smooth muscle cells.
 21. Themethod of claim 20, wherein said detecting is with the antibody specificfor α-SMA.
 22. The method of claim 20, wherein said detecting is withthe antibody specific for calponin.